SMART MODE:

NORMAL GENOMIC

• Scott SP, Weber IT, Harrison RW, Carey J, Tanaka JC
• A functioning chimera of the cyclic nucleotide-binding domain from the bovine retinal rod ion channel and the DNA-binding domain from catabolite gene-activating protein.
• Biochemistry. 2001; 40: 7464-73
• Display abstract

• The eukaryotic cyclic nucleotide-gated (CNG) ion channels are a family of large membrane proteins activated by cytoplasmic cGMP or cAMP. Their cyclic nucleotide-binding domain is structurally homologous with that of the catabolite gene-activator protein (CAP), a soluble Escherichia coli transcription factor. Differences in ligand activation among sensory channels suggest differences in the underlying molecular mechanisms of signal readout. To study the structural, functional, and conformational consequences of nucleotide binding, we fused the cyclic nucleotide-binding domain from the bovine retinal rod CNG channel alpha subunit (Bralpha) to the DNA-binding domain from CAP. The chimera forms a soluble dimer that binds both cGMP and cAMP with association constants of 3.7 x 10(4) M(-1) for [(3)H]cGMP and 3.1 x 10(4) M(-1) for [(3)H]cAMP. The binding of cAMP, but not cGMP, exposes a chymotrypsin cleavage site in the chimera at a position similar to the site in the CAP exposed by cAMP binding. At high cAMP concentrations, a biphasic pattern of cleavage is seen, suggesting that the low-affinity cAMP binding sites are also occupied. Cyclic AMP promotes specific binding to a DNA fragment encoding the lac operator region; the K(d) for the protein-DNA binding is approximately 200 nM, which is 2-fold higher than the K(d) for CAP under identical conditions. A 7 A crystal structure shows that the overall secondary and tertiary structure of Bralpha/CAP is the same as that of CAP with two cAMP molecules bound per dimer. The biochemical characterization of the chimera suggests it will be a useful system for testing hypotheses about channel activation, providing further insight into channel function.

• Cheng X, Phelps C, Taylor SS
• Differential binding of cAMP-dependent protein kinase regulatory subunit isoforms Ialpha and IIbeta to the catalytic subunit.
• J Biol Chem. 2001; 276: 4102-8
• Display abstract

• Limited trypsin digestion of type I cAMP-dependent protein kinase holoenzyme results in a proteolytic-resistant Delta(1-72) regulatory subunit core, indicating that interaction between the regulatory and catalytic subunits extends beyond the autoinhibitory site in the R subunit at the NH(2) terminus. Sequence alignment of the two R subunit isoforms, RI and RII, reveals a significantly sequence diversity at this specific region. To determine whether this sequence diversity is functionally important for interaction with the catalytic subunit, specific mutations, R133A and D328A, are introduced into sites adjacent to the active site cleft in the catalytic subunit. While replacing Arg(133) with Ala decreases binding affinity for RII, interaction between the catalytic subunit and RI is not affected. In contrast, mutant C(D328A) showed a decrease in affinity for binding RI while maintaining similar affinities for RII as compared with the wild-type catalytic subunit. These results suggest that sequence immediately NH(2)-terminal to the consensus inhibition site in RI and RII interacts with different sites at the proximal region of the active site cleft in the catalytic subunit. These isoform-specific differences would dictate a significantly different domain organization in the type I and type II holoenzymes.

• Li F et al.
• Consequences of cAMP and catalytic-subunit binding on the flexibility of the A-kinase regulatory subunit.
• Biochemistry. 2000; 39: 15626-32
• Display abstract

• A combination of site-directed labeling and time-resolved fluorescence anisotropy was used to further elucidate the structure and underlying dynamic features of the type I regulatory (R(I)(alpha)) subunit of the cAMP-dependent protein kinase. Specifically, the consequences of cAMP and the catalytic (C)-subunit binding on the backbone flexibility around seven sites of cysteine substitution and fluorescein maleimide labeling (Thr(6)Cys, Leu(66)Cys, Ser(75)Cys, Ser(81)Cys, Ser(99)Cys, Ser(145)Cys, and Ser(373)Cys) in the R(I)(alpha) subunit were assessed. Focusing on the fast rotational correlation time, the results indicate that most of the interdomain segment connecting the dimerization/docking (D/D) and tandem cAMP-binding domains is probably weakly associated with the latter domain. Also, this segment becomes more tightly bound to the C subunit upon holoenzyme formation. The results also suggest that there is a short 'hinge' segment (around Leu(66)Cys) that could allow the structured interdomain/cAMP-binding and D/D domains to pivot about each other. Finally, cAMP binding dramatically reduces the backbone flexibility around only the two sites of cysteine substitution in the cAMP-binding domains, suggesting a selective structural stabilization caused by cAMP and a "tight" coupling of low-nanosecond fluctuations selectively within the tandem cAMP-binding domains.

• Holcroft CC, Egan SM
• Interdependence of activation at rhaSR by cyclic AMP receptor protein, the RNA polymerase alpha subunit C-terminal domain, and rhaR.
• J Bacteriol. 2000; 182: 6774-82
• Display abstract

• The Escherichia coli rhaSR operon encodes two AraC family transcription activators, RhaS and RhaR, and is activated by RhaR in the presence of L-rhamnose. beta-Galactosidase assays of various rhaS-lacZ promoter fusions combined with mobility shift assays indicated that a cyclic AMP receptor protein (CRP) site located at -111.5 is also required for full activation of rhaSR expression. To address the mechanisms of activation by CRP and the RNA polymerase alpha-subunit C-terminal domain (alpha-CTD) at rhaSR, we tested the effects of alanine substitutions in CRP activating regions 1 and 2, overexpression of a truncated version of alpha (alpha-Delta235), and alanine substitutions throughout alpha-CTD. We found that DNA-contacting residues in alpha-CTD are required for full activation, and for simplicity, we discuss alpha-CTD as a third activator of rhaSR. CRP and RhaR could each partially activate transcription in the absence of the other two activators, and alpha-CTD was not capable of activation alone. In the case of CRP, this suggests that this activation involves neither an alpha-CTD interaction nor cooperative binding with RhaR, while in the case of RhaR, this suggests the likelihood of direct interactions with core RNA polymerase. We also found that CRP, RhaR, and alpha-CTD each have synergistic effects on activation by the others, suggesting direct or indirect interactions among all three. We have some evidence that the alpha-CTD-CRP and alpha-CTD-RhaR interactions might be direct. The magnitude of the synergistic effects was usually greater with just two activators than with all three, suggesting possible redundancies in the mechanisms of activation by CRP, alpha-CTD, and RhaR.

• Bown JA, Kolb A, Meares CF, Ishihama A, Minchin SD, Busby SJ
• Positioning of region 4 of the Escherichia coli RNA polymerase sigma(70) subunit by a transcription activator.
• J Bacteriol. 2000; 182: 2982-4
• Display abstract

• A DNA cleavage reagent, specifically tethered to residue 581 of the Escherichia coli RNA polymerase sigma(70) subunit, has been used to investigate the location of sigma(70) region 4 in different complexes at the galp(1) promoter and the effect of the cyclic AMP receptor protein. The positions of DNA cleavage by the reagent are not affected by the cyclic AMP receptor protein. We conclude that transcription activation at the galp(1) promoter by the cyclic AMP receptor protein does not involve major conformation changes in or repositioning of sigma(70) region 4.

• Canaves JM, Leon DA, Taylor SS
• Consequences of cAMP-binding site mutations on the structural stability of the type I regulatory subunit of cAMP-dependent protein kinase.
• Biochemistry. 2000; 39: 15022-31
• Display abstract

• The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with two tandem cAMP-binding domains, A and B. The importance of cAMP binding on the stability of the R subunit was probed by intrinsic fluorescence and circular dichroism (CD) in the presence and absence of urea. Several mutants were characterized. The site-specific mutants R(R209K) and R(R333K) had defects in cAMP-binding sites A and B, respectively. R(M329W) had an additional tryptophan in domain B. Delta(260-379)R lacked Trp260 and domain B. The most destabilizing mutation was R209K. Both CD and fluorescence experiments carried out in the presence of urea showed a decrease in cooperativity of the unfolding, which also occurred at lower urea concentrations. Unlike native R, R(R209K) was not stabilized by excess cAMP. Additionally, CD revealed significant alterations in the secondary structure of the R209K mutant. Therefore, Arg209 is important not only as a contact site for cAMP binding but also for the intrinsic structural stability of the full-length protein. Introducing the comparable mutation into domain B, R333K, had a smaller effect on the integrity and stability of domain A. Unfolding was still cooperative; the protein was stabilized by excess cAMP, but the unfolding curve was biphasic. The R(M329W) mutant behaved functionally like the native protein. The Delta(260-379)R deletion mutant was not significantly different from wild-type RIalpha in its stability. Consequently, domain B and the interaction between Trp260 and cAMP bound to site A are not critical requirements for the structural stability of the cAPK regulatory subunit.

• Passner JM, Schultz SC, Steitz TA
• Modeling the cAMP-induced allosteric transition using the crystal structure of CAP-cAMP at 2.1 A resolution.
• J Mol Biol. 2000; 304: 847-59
• Display abstract

• After an allosteric transition produced by the binding of cyclic AMP (cAMP), the Escherichia coli catabolite gene activator protein (CAP) binds DNA specifically and activates transcription. The three-dimensional crystal structure of the CAP-cAMP complex has been refined at 2.1 A resolution, thus enabling a better evaluation of the structural basis for CAP phenotypes, the interactions of cAMP with CAP and the roles played by water structure. A review of mutational analysis of CAP together with the additional structural information presented here suggests a possible mechanism for the cAMP-induced allostery required for DNA binding and transcriptional activation. We hypothesize that cAMP binding may reorient the coiled-coil C-helices, which provide most of the dimer interface, thereby altering the relative positions of the DNA-binding domains of the CAP dimer. Additionally, cAMP binding may cause a further rearrangement of the DNA-binding and cAMP-binding domains of CAP via a flap consisting of beta-strands 4 and 5 which lies over the cAMP.

• Mukhopadhyay J, Sur R, Parrack P
• Functional roles of the two cyclic AMP-dependent forms of cyclic AMP receptor protein from Escherichia coli.
• FEBS Lett. 1999; 453: 215-8
• Display abstract

• The cyclic AMP receptor protein activates transcription in Escherichia coli, only when complexed with cyclic AMP. The cyclic AMP receptor protein-cyclic AMP complex formed at low concentrations of cyclic AMP has a different conformation from either cyclic AMP receptor protein alone or its complex with cyclic AMP formed at high cyclic AMP concentrations. Various biophysical data suggest that the latter complex resembles free cyclic AMP receptor protein. We have examined the conformational and biological properties of cyclic AMP receptor protein as a function of cyclic AMP concentrations, using the gal operon of E. coli. A biphasic behavior is observed. It is shown that only the complex formed at lower concentrations of cyclic AMP is the transcriptionally active form. This difference between the complexes at different levels of cyclic AMP arises from a decreased ability of the cyclic AMP receptor protein-cyclic AMP complex at high cyclic AMP concentrations to bind to DNA at specific sites.

• Busby S, Ebright RH
• Transcription activation by catabolite activator protein (CAP).
• J Mol Biol. 1999; 293: 199-213
• Display abstract

• Transcription activation by Escherichia coli catabolite activator protein (CAP) at each of two classes of simple CAP-dependent promoters is understood in structural and mechanistic detail. At class I CAP-dependent promoters, CAP activates transcription from a DNA site located upstream of the DNA site for RNA polymerase holoenzyme (RNAP); at these promoters, transcription activation involves protein-protein interactions between CAP and the RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex. At class II CAP-dependent promoters, CAP activates transcription from a DNA site that overlaps the DNA site for RNAP; at these promoters, transcription activation involves both: (i) protein-protein interactions between CAP and RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex; and (ii) protein-protein interactions between CAP and RNAP alpha subunit N-terminal domain that facilitates isomerization of the RNAP-promoter closed complex to the RNAP-promoter open complex. Straightforward combination of the mechanisms for transcription activation at class I and class II CAP-dependent promoters permits synergistic transcription activation by multiple molecules of CAP, or by CAP and other activators. Interference with determinants of CAP or RNAP involved in transcription activation at class I and class II CAP-dependent promoters permits "anti-activation" by negative regulators. Basic features of transcription activation at class I and class II CAP-dependent promoters appear to be generalizable to other activators.

• Passner JM, Steitz TA
• The structure of a CAP-DNA complex having two cAMP molecules bound to each monomer.
• Proc Natl Acad Sci U S A. 1997; 94: 2843-7
• Display abstract

• The 2.2 A resolution crystal structure of the Escherichia coli catabolite gene activator protein (CAP) complexed with cAMP and a 46-bp DNA fragment reveals a second cAMP molecule bound to each protein monomer. The second cAMP is in the syn conformation and is located on the DNA binding domain interacting with the helix-turn-helix, a beta-hairpin from the regulatory domain and the DNA (via water molecules). The presence of this second cAMP site resolves the apparent discrepancy between the NMR and x-ray data on the conformation of cAMP, and explains the cAMP concentration-dependent behaviors of the protein. In addition, this site's close proximity to mutations affecting transcriptional activation and its water-mediated interactions with a DNA recognition residue (E181) and DNA raise the possibility that this site has biological relevance.

• Dethiollaz S, Eichenberger P, Geiselmann J
• Influence of DNA geometry on transcriptional activation in Escherichia coli.
• EMBO J. 1996; 15: 5449-58
• Display abstract

• Transcription from many Escherichia coli promoters can be activated by the cAMP-CRP complex bound at different locations upstream of the promoter. At some locations the mechanism of activation involves direct protein-protein contacts between CRP and the RNA polymerase. We positioned the CRP binding site at various distances from the transcription start site of the malT promoter and measured the in vivo activities of these promoter variants. From the activation profiles we deduce that the protein-protein interactions involved in transcriptional activation are rather rigid. A heterologous protein (IHF) that bends the DNA to a similar degree as does CRP activates transcription when bound at sites equivalent to activating positions for CRP. DNA geometry makes a major contribution to the process of transcriptional activation and DNA upstream of the activator binding site participates in this process. Removal of this DNA decreases the capacity of the malT promoter to be activated by CRP in vitro. We conclude that both DNA topology and direct protein-protein contacts contribute to transcriptional activation and that the relative importance of these two modes of activation depends on the nature of the activator and on the location of the activator binding site.

• Herberg FW, Taylor SS, Dostmann WR
• Active site mutations define the pathway for the cooperative activation of cAMP-dependent protein kinase.
• Biochemistry. 1996; 35: 2934-42
• Display abstract

• cAMP-dependent protein kinase (cAPK) is a heterotetramer containing two regulatory (R) and two catalytic (C) subunits. Each R-subunit contains two tandem cAMP-binding domains, and activation of cAPK is mediated by the cooperative, high affinity binding of cAMP to these two domains. Mutant R-subunits containing one intact high affinity cAMP-binding site and one defective site were used to define the pathway for activation and to delineate the unique roles that each cAMP-binding domain plays. Two mutations were introduced by replacing the essential Arg in each cAMP-binding site with Lys (R209K in Site A and R333K in Site B). Also, the double mutant (R209/333K) was constructed. Analysis of cAMP binding and dissociation and the apparent constants for holoenzyme activation and R- and C-subunit interaction, measured by analytical gel filtration and surface plasmon resonance, established the following: (1) For rR(R209K), occupancy of Site B is not sufficient to activate the holoenzyme; the low affinity Site A must also be occupied. In rR(R333K), Site A retains its high affinity for cAMP, but Site A cannot bind until the low affinity Site B is occupied. Thus, both mutants, for different reasons, have similar Ka's for activation that are approximately 20-fold higher than that of the wild-type holoenzyme. The double mutant with two defective sites is no worse than either single mutant. (2) Kinetic analysis of cAMP binding showed that the mutation in Site A or B abolishes high affinity cAMP binding to that site and slightly weakens the affinity of the adjacent site for cAMP. (3) In the presence of MgATP, both mutants rapidly form a stable holoenzyme even in the presence of cAMP in contrast to the wild-type R where holoenzyme forms slowly in vitro and requires dialysis. Regarding the mechanism of activation based on these and other mutants and from kinetic data, the following conclusions are reached: Site A provides the major contact site with the C-subunit; Site B is not essential for holoenzyme formation. Occupancy of Site A by cAMP mediates dissociation of the C-subunit. Site A is inaccessible to cAMP in the full length holoenzyme, while Site B is fully accessible. Access of cAMP to Site A is mediated by Site B. Thus Site B not only helps to shield Site A, it also provides the specific signal that "opens up" Site A. Finally, a nonfunctional Site A in the holoenzyme prevents stable binding of cAMP to Site B in the absence of subunit dissociation.

• Niu W, Kim Y, Tau G, Heyduk T, Ebright RH
• Transcription activation at class II CAP-dependent promoters: two interactions between CAP and RNA polymerase.
• Cell. 1996; 87: 1123-34
• Display abstract

• At Class II catabolite activator protein (CAP)-dependent promoters, CAP activates transcription from a DNA site overlapping the DNA site for RNA polymerase. We show that transcription activation at Class II CAP-dependent promoters requires not only the previously characterized interaction between an activating region of CAP and the RNA polymerase alpha subunit C-terminal domain, but also an interaction between a second, promoter-class-specific activating region of CAP and the RNA polymerase alpha subunit N-terminal domain. We further show that the two interactions affect different steps in transcription initiation. Transcription activation at Class II CAP-dependent promoters provides a paradigm for understanding how an activator can make multiple interactions with the transcription machinery, each interaction being responsible for a specific mechanistic consequence.

• Steinberg RA, Symcox MM, Sollid S, Ogreid D
• Arginine 210 is not a critical residue for the allosteric interactions mediated by binding of cyclic AMP to site A of regulatory (RIalpha) subunit of cyclic AMP-dependent protein kinase.
• J Biol Chem. 1996; 271: 27630-6
• Display abstract

• The guanidinium groups of conserved arginines in the two intrachain cAMP-binding sites of regulatory (R) subunit of cAMP-dependent protein kinase have been implicated in the allosteric interactions by which cAMP binding leads to kinase activation. We have investigated the functional role of Arg-210, the conserved arginine in site A of murine type Ialpha R subunit, by analyzing the effects of nine different substitutions at this residue on cAMP binding and allosteric properties of bacterially expressed RIalpha subunits. All substitutions reduced the cAMP binding affinity of site A, but the magnitude of reduction varied from several hundredfold to 10(6)-fold. The differential effects of the different substitutions could not easily be rationalized by interactions with cAMP and might, in part, reflect interactions with other residues in the unoccupied cAMP-binding pocket. None of the Arg-210 substitutions appeared to disrupt the allosteric interaction by which occupation of site A slows dissociation of cAMP from site B, although the effect was difficult to elicit in full with mutations that had strong effects on cAMP binding. The two weakest substitutions, Arg-210 --> Ile and Arg-210 --> Thr, could be shown to have essentially no effect on the allosteric interaction by which occupation of site A reduces the affinity of R subunit for the catalytic subunit. The weaker mutations had a smaller effect on kinase activation by the suboptimal activator Rp-adenosine cyclic 3',5'-phosphorothioate than by cAMP, suggesting that the analog largely bypasses interactions with the guanidinium group of Arg-210.

• Adhya S, Ryu S, Garges S
• Role of allosteric changes in cyclic AMP receptor protein function.
• Subcell Biochem. 1995; 24: 303-21

• Tang H, Severinov K, Goldfarb A, Fenyo D, Chait B, Ebright RH
• Location, structure, and function of the target of a transcriptional activator protein.
• Genes Dev. 1994; 8: 3058-67
• Display abstract

• We have isolated and characterized single-amino-acid substitution mutants of RNA polymerase alpha subunit defective in CAP-dependent transcription at the lac promoter but not defective in CAP-independent transcription. Our results establish that (1) amino acids 258-265 of alpha constitute an "activation target" essential for CAP-dependent transcription at the lac promoter but not essential for CAP-independent transcription, (2) amino acid 261 is the most critical amino acid of the activation target, (3) amino acid 261 is distinct from the determinants for alpha-DNA interaction, and (4) the activation target may fold as a surface amphipathic alpha-helix. We propose a model for transcriptional activation at the lac promoter that integrates these and other recent results regarding transcriptional activation and RNA polymerase structure and function.

• Cox S, Taylor SS
• Holoenzyme interaction sites in the cAMP-dependent protein kinase. Histidine 87 in the catalytic subunit complements serine 99 in the type I regulatory subunit.
• J Biol Chem. 1994; 269: 22614-22
• Display abstract

• Two mutations of the catalytic (C) subunit of the cAMP-dependent protein kinase where His87 was changed to Ala and Asp were expressed in Escherichia coli and purified. These mutants were phosphorylated at Thr197 and were catalytically active, although some changes in their kinetic parameters were observed. The most striking differences were in their interaction with the physiological inhibitors. Both mutants were inhibited by protein kinase inhibitor with Ki values below 50 nM. Both mutants were defective in their interaction with the type I regulatory (RI) subunit as measured by (i) the rate of holoenzyme formation with cAMP bound RI-subunit and (ii) the apparent Kd with the cAMP-free RI-subunit. The rate of holoenzyme formation was impaired both in the presence and absence of ATP with the His to Asp mutant showing the greatest effect. The mutant C-subunits were also combined with RI-subunits that contained mutations in the autoinhibitor sequence at Arg94 (P-3) and Ser99 (P + 2). Complementarity between His87 and Ser99 was established, but not between His87 and Arg94. Holoenzyme formation with a Ser99-->Lys mutant RI-subunit was less dependent on ATP when combined with either of the C-subunit mutants than when it was combined with the wild-type C-subunit. The apparent Kd values in the presence of ATP for the mutant combinations were also measured. The Ser99-->Lys mutant was compensated for by both His87 mutants. The His87-->Ala C-subunit mutant was unable to form an inhibited holoenzyme complex with a mutant RI-subunit which was defective in cAMP binding to the A-site. This indicated that this R-subunit was defective in C-subunit recognition as well as in cAMP binding. The roles of His87 on the C-subunit and Ser99 and Arg209 on the RI-subunit in R-C interactions are discussed.

• Symcox MM, Cauthron RD, Ogreid D, Steinberg RA
• Arg-242 is necessary for allosteric coupling of cyclic AMP-binding sites A and B of RI subunit of cyclic AMP-dependent protein kinase.
• J Biol Chem. 1994; 269: 23025-31
• Display abstract

• The functional consequences of Arg-242 to Ser or Lys substitutions in type I alpha regulatory (R) subunits of cAMP-dependent protein kinase were analyzed by using recombinant murine R subunits expressed in Escherichia coli. These mutations arose in cAMP-resistant mutants to S49 mouse lymphoma cells and were shown previously to inhibit cAMP binding to site A, the more amino-terminal of two intrachain cAMP-binding sites. Binding of cAMP to site A of the mutant R subunits could be detected by cAMP-dependent quenching of endogenous tryptophan fluorescence, [3H]cAMP binding to mutant R subunits with the Arg-242 mutations without or with an inactivating mutation in site B, or biphasic dissociation of [3H]cAMP from the mutant subunits at low temperature. The mutations reduced site A affinities by about 25-fold, and the reductions were attributable to accelerated rates of cAMP dissociation. While the presence of cAMP in site A retards dissociation of [3H]cAMP from site B of wild-type R subunits, saturation of site A had little or no effect on dissociation of [3H]cAMP from site B of the mutant subunits. The predominant effect of the mutations, therefore, was loss of allosteric coupling between the two cAMP-binding sites. A second allosteric interaction, that coupling occupation of site A with a reduced affinity of R for catalytic subunit, was inhibited only partially by these mutations at Arg-242.

• Zhou Y, Pendergrast PS, Bell A, Williams R, Busby S, Ebright RH
• The functional subunit of a dimeric transcription activator protein depends on promoter architecture.
• EMBO J. 1994; 13: 4549-57
• Display abstract

• In Class I CAP-dependent promoters, the DNA site for CAP is located upstream of the DNA site for RNA polymerase. In Class II CAP-dependent promoters, the DNA site for CAP overlaps the DNA site for RNA polymerase, replacing the -35 site. We have used an 'oriented heterodimers' approach to identify the functional subunit of CAP at two Class I promoters having different distances between the DNA sites for CAP and RNA polymerase [CC(-61.5) and CC(-72.5)] and at one Class II promoter [CC(-41.5)]. Our results indicate that transcription activation at Class I promoters, irrespective of the distance between the DNA sites for CAP and RNA polymerase, requires the activating region of the promoter-proximal subunit of CAP. In striking contrast, our results indicate that transcription activation at Class II promoters requires the activating region of the promoter-distal subunit of CAP.

• Herberg FW, Dostmann WR, Zorn M, Davis SJ, Taylor SS
• Crosstalk between domains in the regulatory subunit of cAMP-dependent protein kinase: influence of amino terminus on cAMP binding and holoenzyme formation.
• Biochemistry. 1994; 33: 7485-94
• Display abstract

• The regulatory (R) subunit of cAMP-dependent protein kinase os an asymmetric multidomain protein with a dimerization domain at the N-terminus, an autoinhibitors site, and two cAMP binding domains at the C-terminus. Activation of the tetrameric holoenzyme is mediated by the cooperative binding of cAMP to the two cAMP binding sites. To better understand how the various domains influence each other, the N-terminus (delta 1-91) up to the autoinhibitor site was deleted. Not only did this monomeric deletion mutant, purified from Escherichia coli, still bind cAMP and the catalytic (C) subunit with high affinity, holoenzyme formation was actually accelerated by at least 50-fold. MgATP also was not required for rapid reassociation of (delta 1-91)R(cAMP)2 and C. The Kd(cAMP) and the Ka(cAMP) were similar to those for holoenzyme formed with full-length R; however, cooperatively was lost. Thus the N-terminus, either by inter- or intraprotomer contacts, not only impedes holoenzyme formation but also influences the cooperative binding of cAMP. The 1-91 deletion also renders the remaining fragment resistant to proteolytic degradation. Finally, unlike full-length R, the mutant protein can migrate freely into the nucleus. Surface plasmon resonance studies for the first time enabled direct measurements of the association and dissociation rate constants both for the intact R and for (delta 1-91)R. Both displayed very fast on-rates (1 x 10(-5) M-1 s-1 and 1.1 x 10(-5) M-1 s-1, respectively) and extremely slow off-rates (2.3 x 10(5) M-1 and 4.3 x 10(5) M-1, respectively). Thus, unlike the heat-stable protein kinase inhibitor, the region preceding the autoinhibitor site in R does not contribute in a quantitatively significant way to the high-affinity binding of C.

• Niu W, Zhou Y, Dong Q, Ebright YW, Ebright RH
• Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP). I. Saturation and alanine-scanning mutagenesis.
• J Mol Biol. 1994; 243: 595-602
• Display abstract

• It has been proposed that the surface loop consisting of amino acid residues 152 to 166 of the catabolite gene activator protein (CAP) of Escherichia coli makes direct protein-protein contact with RNA polymerase at the lac promoter. In this work, we have used targeted saturation mutagenesis of codons 152 to 166 of the gene encoding CAP, followed by a screen, to isolate more than 200 independent mutants of CAP defective in transcription activation but not defective in DNA binding. All isolated single-substitution mutants map to just eight amino acid residues; 156, 157, 158, 159, 160, 162, 163 and 164. We propose that these residues define the full extent of the epitope on CAP for the proposed CAP-RNA polymerase interaction. In addition, we have constructed alanine substitutions at each position from residue 152 to 166 of CAP, and we have analyzed the effects on transcription activation at the lac promoter and on DNA binding. Alanine substitution of Thr158 results in an approximately eightfold specific defect in transcription activation. In contrast, alanine substitution of no other residue tested results in a more than twofold specific defect in transcription activation. We conclude that, for Thr158, side-chain atoms beyond C beta are essential for transcription activation at the lac promoter, and we propose that Thr158 OH7 gamma makes direct contact with RNA polymerase in the ternary complex of lac promoter, CAP and RNA polymerase. We conclude further that for no residue other than Thr158 are side-chain atoms beyond C beta essential for transcription activation at the lac promoter.

• Zhou Y, Merkel TJ, Ebright RH
• Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP). II. Role at Class I and class II CAP-dependent promoters.
• J Mol Biol. 1994; 243: 603-10
• Display abstract

• CAP-dependent promoters can be divided into classes based on the position of the DNA site for CAP. In class I CAP-dependent promoters, the DNA site for CAP is located upstream of the DNA site for polymerase; the DNA site for CAP can be located at various distances from the transcription start point, provided that the DNS site for CAP and the DNA site for RNA polymerase are on the same face of the DNA helix. In class II CAP-dependent promoters, the DNA site for CAP overlaps the DNA site for RNA polymerase, replacing the -35 determinants for binding of RNA polymerase. In previous work, we have shown that a surface loop consisting of amino acid residues 152 to 166 of CAP is essential for transcription activation at the best-characterized class I CAP-dependent promoter, the lac promoter, and we proposed that this surface loop makes direct protein-protein contact with RNA polymerase in the ternary complex of lac promoter, CAP, and RNA polymerase. Here, we show that the surface loop consisting of amino acid residues 152 to 166 is essential for transcription activation at other class I CAP-dependent promoters and at a class II CAP-dependent promoter. We show further that the effects of alanine substitutions of residues 152 to 166 are qualitatively identical at the lac promoter and other class I CAP-dependent promoters, but are different at a class II CAP-dependent promoter. We propose that the surface loop consisting of residues 152 to 166 makes identical molecular interactions in transcription activation at all class I CAP-dependent promoters, irrespective of distance between the DNA site for CAP and the transcription start point, but makes a different set of molecular interactions in transcription activation at class II CAP-dependent promoters.

• Perez-Martin J, Espinosa M
• Protein-induced bending as a transcriptional switch.
• Science. 1993; 260: 805-7
• Display abstract

• The question of whether protein-induced DNA bending can act as a switch factor when placed upstream of an array of promoters located in tandem was investigated in vivo. The catabolite activating protein binding site of the fur operon was replaced by the binding site of the RepA repressor protein, which is able to bend DNA immediately after binding. Appropriately phased induced bending could act as a transcriptional switch factor in vivo.

• Kolb A, Busby S, Buc H, Garges S, Adhya S
• Transcriptional regulation by cAMP and its receptor protein.
• Annu Rev Biochem. 1993; 62: 749-95

• Breul A, Assmann H, Golz R, von Wilcken-Bergmann B, Muller-Hill B
• Mutants with substitutions for Glu171 in the catabolite activator protein (CAP) of Escherichia coli activate transcription from the lac promoter.
• Mol Gen Genet. 1993; 238: 155-60
• Display abstract

• Single amino acid substitutions for residue Glu171 in helix E of the catabolite gene activator protein (CAP) of Escherichia coli have been reported to abolish activation of transcription without impairing binding to the CAP site of the lac promoter. The negative charge of Glu171 was proposed to transmit the activating signal from CAP to RNA polymerase. However, this idea has been challenged by later work. We set up a system to re-examine this issue. We analysed the ability of mutant CAP-E171L and CAP-E171K proteins to bind a near-consensus CAP site in vivo and found it to be diminished fourfold relative to wild type in each case. Activation of lac transcription by these mutant proteins remains the same as with wild-type CAP. Thus our results confirm that Glu171 in helix E of CAP is not involved directly in the activation of transcription. Yet CAP-E171K does not activate transcription as well as wild-type CAP under all circumstances. Possible reasons for this absence of activation are discussed.

• Zhou Y, Zhang X, Ebright RH
• Identification of the activating region of catabolite gene activator protein (CAP): isolation and characterization of mutants of CAP specifically defective in transcription activation.
• Proc Natl Acad Sci U S A. 1993; 90: 6081-5
• Display abstract

• We have isolated 21 mutants of catabolite gene activator protein (CAP) defective in transcription activation at the lac promoter but not defective in DNA binding. The amino acid substitutions in the mutants map to a single region of CAP: amino acids 156-162. As assessed in vitro, the substituted CAP variants are nearly completely unable to activate transcription at the lac promoter but bind to DNA with the same affinity and bend DNA to the same extent as wild-type CAP. Our results establish that amino acids 156-162 are critical for transcription activation at the lac promoter but not for DNA binding and DNA bending. In the structure of CAP, amino acids 156-162 are part of a surface loop. We propose that this surface loop makes a direct protein-protein contact with RNA polymerase at the lac promoter.

• Ebright RH
• Transcription activation at Class I CAP-dependent promoters.
• Mol Microbiol. 1993; 8: 797-802
• Display abstract

• Catabolite gene activator protein (CAP)-dependent promoters can be grouped into three classes, based on the requirement for transcription activation and the position of the DNA site for CAP. Class I CAP-dependent promoters require only CAP for transcription activation and have the DNA site for CAP located upstream of the DNA site for RNA polymerase. Amino acids 156 to 162 of the promoter-proximal subunit of CAP are essential for transcription activation at Class I CAP-dependent promoters, but are not essential for DNA binding, and are not essential for DNA bending. In the structure of the CAP-DNA complex, these amino acids are located in a surface loop and form a cluster on the surface of the CAP-DNA complex. Amino acids 261, 265, and 270 of the alpha subunit of RNA polymerase are essential for response to transcription activation by CAP at Class I CAP-dependent promoters. Several lines of evidence indicate that transcription activation at Class I CAP-dependent promoters requires a direct protein-protein contact between amino acids 156 to 162 of the promoter-proximal subunit of CAP and a molecule of RNA polymerase bound adjacent to CAP on the same face of the DNA helix. It is a strong possibility that this direct protein-protein contact involves amino acids 261 and 265 of the alpha subunit of RNA polymerase.

• Johnson DA, Leathers VL, Martinez AM, Walsh DA, Fletcher WH
• Fluorescence resonance energy transfer within a heterochromatic cAMP-dependent protein kinase holoenzyme under equilibrium conditions: new insights into the conformational changes that result in cAMP-dependent activation.
• Biochemistry. 1993; 32: 6402-10
• Display abstract

• Previous studies of the ligand regulation of the cAMP-dependent protein kinase have demonstrated the cAMP-mediated dissociation of the holoenzyme by using nonequilibrium techniques; i.e., gel filtration, ion-exchange chromatography, and differential centrifugation. While physically mild, these could have caused weakly associated species to dissociate, thereby providing a potentially flawed interpretation of the mechanism of activation of the protein kinase. To assess this, the activation of the cAMP-dependent protein kinase has been monitored under equilibrium conditions using dipolar fluorescence energy transfer to measure changes in the proximity relations between the catalytic (C) and regulatory (R) subunits that compose the holoenzyme. Specifically, we prepared a heterochromatically labeled protein kinase type II holoenzyme, with the regulatory and catalytic subunits labeled with sulforhodamine and carboxyfluorescein, respectively, and monitored the exchange of electronic excitation energy between the C and R subunits by both donor lifetime and steady-state fluorescence. Biochemically, the heterochromatic holoenzyme was closely identical to the native protein with regard to cAMP-induced increase in catalytic activity, reassociation of C and R subunits, inhibition of catalytic activity by the specific protein kinase inhibitor (PKI), and observed dissociation examined by gel filtration upon cAMP addition. However, under equilibrium conditions, the energy-transfer measurements revealed that the addition of cAMP to this heterochromatic reporter complex promoted an estimated 10-A increase in the distance between the derivatization sites on C and R but not a dissociation of these subunits. Addition of PKI plus cAMP promoted full dissociation of the two subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

• Heyduk T, Lee JC, Ebright YW, Blatter EE, Zhou Y, Ebright RH
• CAP interacts with RNA polymerase in solution in the absence of promoter DNA.
• Nature. 1993; 364: 548-9
• Display abstract

• Protein-protein interactions between transcription activator proteins and RNA polymerase or basal transcription factors have been suggested to be important for transcription activation. Interactions between catabolite gene activator protein (CAP) and RNA polymerase have been proposed based on face-of-helix-dependent transcription activation by CAP and based on face-of-helix-dependent cooperative binding of CAP and RNA polymerase to promoter DNA. Mutants of CAP specifically defective in transcription activation have been isolated (mutants defective in transcription activation, but not defective in DNA binding and DNA bending). All such mutants contain amino-acid substitutions within a surface loop consisting of amino acids 152 to 166 of CAP. Here we use the thermodynamically rigorous technique of fluorescence polarization to show that CAP interacts with RNA polymerase in solution in the absence of promoter DNA (KD,app = 2.8 x 10(-7) M), whereas [Ala158]CAP, a mutant of CAP specifically defective in transcription activation, does not.

• Dong Q, Ebright RH
• DNA binding specificity and sequence of Xanthomonas campestris catabolite gene activator protein-like protein.
• J Bacteriol. 1992; 174: 5457-61
• Display abstract

• The Xanthomonas campestris catabolite gene activator protein-like protein (CLP) can substitute for the Escherichia coli catabolite gene activator protein (CAP) in transcription activation at the lac promoter (V. de Crecy-Lagard, P. Glaser, P. Lejeune, O. Sismeiro, C. Barber, M. Daniels, and A. Danchin, J. Bacteriol. 172:5877-5883, 1990). We show that CLP has the same DNA binding specificity as CAP at positions 5, 6, and 7 of the DNA half site. In addition, we show that the amino acids at positions 1 and 2 of the recognition helix of CLP are identical to the amino acids at positions 1 and 2 of the recognition helix of CAP:i.e., Arg at position 1 and Glu at position 2.

• Reznikoff WS
• The lactose operon-controlling elements: a complex paradigm.
• Mol Microbiol. 1992; 6: 2419-22
• Display abstract

• The lactose-controlling elements have been considered to be the simple paradigm of a cis-acting genetic regulatory system, containing a promoter whose activity is modulated by an operator and a catabolite gene activator protein (CAP)-binding site. The reality is considerably more complex. We now know that transcription is negatively regulated as a result of the repressor binding to three binding sites: the operator, a secondary repressor-binding site within the lacZ gene and a tertiary repressor-binding site upstream near lacI. In addition to the promoter, the lac-controlling elements contain five promoter-like elements. The physiological role, if any, of these promoter-like elements is not clear, although three of them can be activated by single base pair changes to give high levels of in vivo expression. Finally, the positive activator protein CAP has been found to bind to a secondary site which is coincident with the operator. No role has been identified for this secondary CAP-DNA complex.

• Ebright YW, Chen Y, Pendergrast PS, Ebright RH
• Incorporation of an EDTA-metal complex at a rationally selected site within a protein: application to EDTA-iron DNA affinity cleaving with catabolite gene activator protein (CAP) and Cro.
• Biochemistry. 1992; 31: 10664-70
• Display abstract

• We have developed a simple procedure to incorporate an EDTA-metal complex at a rationally selected site within a full-length protein. Our procedure has two steps: In step 1, we use site-directed mutagenesis to introduce a unique solvent-accessible cysteine residue at the site of interest. In step 2, we derivatize the resulting protein with S-(2-pyridylthio)cysteaminyl-EDTA-metal, a novel aromatic disulfide derivative of EDTA-metal. We have used this procedure to incorporate an EDTA-iron complex at amino acid 2 of the helix-turn-helix motif of each of two helix-turn-helix motif sequence-specific DNA binding proteins, catabolite gene activator protein (CAP) and Cro, and we have analyzed EDTA-iron-mediated DNA affinity cleavage by the resulting protein derivatives. The CAP derivative cleaves DNA at base pair 2 of the DNA half-site in the protein-DNA complex, and the Cro derivative cleaves DNA at base pairs -3 to 5 of the DNA half-site in the protein-DNA complex. We infer that amino acid 2 of the helix-turn-helix motif of CAP is close to base pair 2 of the DNA half-site in the CAP-DNA complex in solution and that amino acid 2 of the helix-turn-helix motif of Cro is close to base pairs -3 to 5 of the DNA half-site in the Cro-DNA complex in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

• Gunasekera A, Ebright YW, Ebright RH
• DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein.
• J Biol Chem. 1992; 267: 14713-20
• Display abstract

• The consensus DNA site for binding of the Escherichia coli catabolite gene activator protein (CAP) is 22 base pairs in length and is 2-fold symmetric: 5'-AAATGTGATCTAGATCACATTT-3'. Positions 4 to 8 of each half of the consensus DNA half-site are the most strongly conserved. In this report, we analyze the effects of substitution of DNA base pairs at positions 4 to 8, the effects of substitution of thymine by uracil and by 5-methylcytosine at positions 4, 6, and 8, and the effect of dam methylation of the 5'-GATC-3' sequence at positions 7 to 10. All DNA sites having substitutions of DNA base pairs at positions 4 to 8 exhibit lower affinities for CAP than does the consensus DNA site, consistent with the proposal that the consensus DNA site is the ideal DNA site for CAP. Specificity for T:A at position 4 appears to be determined solely by the thymine 5-methyl group. Specificity for T:A at position 6 and specificity for A:T at position 8 appear to be determined in part, but not solely, by the thymine 5-methyl group. dam methylation has little effect on CAP.DNA complex formation. The thermodynamically defined consensus DNA site spans 28 base pairs. All, or nearly all, DNA determinants required for maximal affinity for CAP and for maximal thermodynamically defined CAP.DNA ion pair formation are contained within a 28-base pair DNA fragment that has the 22-base pair consensus DNA site at its center. The quantitative data in this report provide base-line thermodynamic data required for detailed investigations of amino acid-base pair and amino acid-phosphate contacts in this protein-DNA complex.

• Zhang X, Zhou Y, Ebright YW, Ebright RH
• Catabolite gene activator protein (CAP) is not an "acidic activating region" transcription activator protein. Negatively charged amino acids of CAP that are solvent-accessible in the CAP-DNA complex play no role in transcription activation at the lac promoter.
• J Biol Chem. 1992; 267: 8136-9
• Display abstract

• It has been suggested that the catabolite gene activator protein (CAP) uses an "acidic activating region" transcription activation mechanism and that Glu171 of CAP is the critical amino acid of the "acidic activating region" of CAP (Irwin, N., and Ptashne, M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8315-8319). In this paper, we show, contrary to the previously published report, that substitution of Glu171 of CAP fails to result in a specific defect in transcription activation at the lac promoter. Furthermore, in this paper, we show that substitution of each other negatively charged amino acid of CAP that is solvent-accessible in the CAP-DNA complex fails to result in a specific defect in transcription activation at the lac promoter. We conclude that CAP does not use an acidic activating region transcription activation mechanism in transcription activation at the lac promoter.

• Wang YH, Scott JD, McKnight GS, Krebs EG
• A constitutively active holoenzyme form of the cAMP-dependent protein kinase.
• Proc Natl Acad Sci U S A. 1991; 88: 2446-50
• Display abstract

• The major function of the regulatory (R) subunit of the cAMP-dependent protein kinase is to bind tightly to the catalytic (C) subunit to form an inactive holoenzyme in the absence of cAMP. The hinge region of the R subunit resembles the substrate recognition site for the C subunit and is known to be involved in the R.C subunit interaction. Two arginine residues in this region, Arg-92 and Arg-93, are suggested to be essential for holoenzyme formation. In this study, a mutant in which Arg-92 and Arg-93 of type II regulatory subunit (RII) were replaced with alanine was constructed. Formation of the holoenzyme from mutant RII and C subunits was analyzed by gel-filtration and cation-exchange chromatography. Mutant RII in its cAMP-free form formed a stable holoenzyme with the C subunit, which dissociated in the presence of cAMP. Interestingly, the holoenzyme formed from mutant RII and C subunits retained full enzymatic activity even in the absence of cAMP. Although mutant RII could no longer be phosphorylated by the C subunit, the rate of [3H]cAMP release from mutant RII.cAMP was increased by addition of the C subunit, indicating that C-induced cAMP release is not the result of the interaction of the C subunit with the hinge region. These results demonstrate that Arg-92 and Arg-93 are not essential for holoenzyme formation but are critical for inhibiting kinase activity in the holoenzyme probably by occupying the substrate binding site. The results suggest that, in addition to the hinge region, a second site on the RII subunit may interact with the C subunit in a cAMP-dependent manner.

• Buechler YJ, Taylor SS
• Mutations in the autoinhibitor site of the regulatory subunit of cAMP-dependent protein kinase I. Replacement of Ala-97 and Ser-99 interferes with reassociation with the catalytic subunit.
• J Biol Chem. 1991; 266: 3491-7
• Display abstract

• Each regulatory (R) subunit of cAMP-dependent protein kinase contains an autoinhibitor site that lies approximately 90-100 residues from the amino terminus. In order to study the importance of this autoinhibitor site in the type I R-subunit for interacting with the catalytic (C) subunit, recombinant techniques were used to replace Ala-97 with Gln, His, Lys, and Arg and to replace Ser-99 with Gly and Lys. All of the mutant proteins having a replacement at Ala-97 showed reduced affinity for the C-subunit ranging from 14- to 55-fold. In general, the decrease in affinity of the Ala-97 mutants for the C-subunit correlated with the increase in size of the side chain. In contrast to wild type R-subunit, where MgATP facilitates holoenzyme formation, MgATP inhibits the reassociation in all of the Ala-97 mutants suggesting that the larger side chains sterically interfere with bound MgATP in the active site of the C-subunit. Whereas MgATP slowed holoenzyme formation, AMP actually accelerated the reassociation of the A97K, A97H (pH 6.0), and A97Q mutants with the C-subunit. Therefore, the side chains of Lys-97, His-97, and Gln-97 can interact either electrostatically or by hydrogen bonding with the phosphate of AMP. This interpretation is reinforced by the fact that the stimulatory effect of AMP on the A97H mutant was pH-dependent. The affinities of the S99G and S99K mutants for the C-subunit were reduced 7- and 24-fold, respectively, suggesting that Ser-99 also may contribute to interactions between the R- and C-subunits.

• Steinberg RA, Gorman KB, Ogreid D, Doskeland SO, Weber IT
• Mutations that alter the charge of type I regulatory subunit and modify activation properties of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells.
• J Biol Chem. 1991; 266: 3547-53
• Display abstract

• Mutations in regulatory (R) subunit of cAMP-dependent protein kinase were analyzed from cAMP-resistant mutants of S49 mouse lymphoma cells by direct sequencing of amplified regions of mutant R subunit cDNAs. Eight distinct single base-change lesions were identified in 24 independent mutants that were hemizygous for expression of mutant R subunits with altered protein charge. CG----TA transitions predominated, but AT----GC transitions and GC----TA transversions were also observed. Four of five spontaneous mutants had identical C----T transitions at CG causing substitution of Trp for Arg-334. Sites mutated in isolates obtained after mutagenesis with ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine were more varied. Six of the lesions (two in binding site A and four in site B) were at amino acid residues that are highly conserved among cAMP-binding sites of R subunits and the Escherichia coli catabolite activator protein. These mutations all either prevented or strongly hindered binding of cyclic nucleotides to the mutated site. One of the remaining lesions (at Arg-242) also prevented cyclic nucleotide binding to the mutated binding site; the other (at Gly-170) had only minimal effects on binding of cyclic nucleotides but, nevertheless, increased the apparent constant for cAMP-dependent kinase activation. These results are discussed with reference to a model for the cAMP-binding sites of R subunit based on the crystal structure of the E. coli catabolite activator protein.

• Zhang XP, Gunasekera A, Ebright YW, Ebright RH
• Derivatives of CAP having no solvent-accessible cysteine residues, or having a unique solvent-accessible cysteine residue at amino acid 2 of the helix-turn-helix motif.
• J Biomol Struct Dyn. 1991; 9: 463-73
• Display abstract

• The Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein. CAP contains a unique solvent-accessible cysteine residue at amino acid 10 of the helix-turn-helix motif. In published work, we have constructed a prototype semi-synthetic site-specific DNA cleavage agent from CAP by use of cysteine-specific chemical modification to incorporate a nucleolytic chelator-metal complex at amino acid 10 of the helix-turn-helix motif [Ebright, R., Ebright, Y., Pendergrast, P.S. and Gunasekera, A., Proc. Natl. Acad. Sci. USA 87, 2882-2886 (1990)]. Construction of second-generation semi-synthetic site-specific DNA cleavage agents from CAP requires the construction of derivatives of CAP having unique solvent-accessible cysteine residues at sites within CAP other than amino acid 10 of the helix-turn-helix motif. In the present work, we have constructed and characterized two derivatives of CAP having no solvent-accessible cysteine residues: [Ser178]CAP and [Leu178]CAP. In addition, in the present work, we have constructed and characterized one derivative of CAP having a unique solvent-accessible cysteine residue at amino acid 2 of the helix-turn-helix motif: [Cys170;Ser178]CAP.

• Jones KW, Shapero MH, Chevrette M, Fournier RE
• Subtractive hybridization cloning of a tissue-specific extinguisher: TSE1 encodes a regulatory subunit of protein kinase A.
• Cell. 1991; 66: 861-72
• Display abstract

• Tissue-specific extinguisher 1 (TSE1) is a trans-acting locus on human chromosome 17 that down-regulates expression of seven liver genes in hepatoma x fibroblast hybrids. To study the mechanism by which TSE1 functions, we used subtractive cDNA hybridization to clone transcripts encoded within a 2-4 Mb segment of chromosome 17 that includes TSE1. High resolution mapping within this region indicated that 8 of 9 different human cDNAs so obtained were distinct from TSE1. The remaining cDNA clone mapped concordantly with TSE1 in a panel of fragment-containing hybrids. DNA sequencing indicated that this cDNA encoded regulatory subunit RI alpha of cAMP-dependent protein kinase, and RI alpha mRNA levels correlated with TSE1 activity in various hybrid lines. Stable transfection of wild-type or cAMP-binding mutant RI alpha alleles into hepatoma recipients produced an extinction phenotype indistinguishable from that encoded by human TSE1. We conclude that TSE1 encodes a regulatory subunit of protein kinase A whose activity differs in different cell types.

• Shabb JB, Buzzeo BD, Ng L, Corbin JD
• Mutating protein kinase cAMP-binding sites into cGMP-binding sites. Mechanism of cGMP selectivity.
• J Biol Chem. 1991; 266: 24320-6
• Display abstract

• The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels.

• Carr DW et al.
• Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif.
• J Biol Chem. 1991; 266: 14188-92
• Display abstract

• The type II cAMP-dependent protein kinase is localized to specific subcellular environments through the binding of the regulatory subunit (RII) dimer to RII-anchoring proteins. Computer-aided analysis of secondary structure, performed on four RII-anchoring protein sequences (the microtubule-associated protein 2, P150, and two thyroid proteins Ht 21 and Ht 31), has identified common regions of approximately 14 residues which display high probabilities of forming amphipathic helices. The potential amphipathic helix region of Ht 31 (Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile) lies between residues 494 and 507. A bacterially expressed 318-amino acid fragment, Ht 31 (418-736), containing the amphipathic helix region, was able to bind RII alpha. Site-directed mutagenesis designed to disrupt the secondary structure in the putative binding helix reduced binding dramatically. Specifically, substitution of proline for Ala-498 significantly diminished RII alpha binding, and similar mutation of Ile-502 or Ile-507 abolished interaction. Mutation of Ala-522 to proline, which is located outside the predicted amphipathic helix region, had no effect on RII alpha binding. These data suggest that anchoring proteins interact with RII alpha via an amphipathic helix binding motif.

• Cadd GG, Uhler MD, McKnight GS
• Holoenzymes of cAMP-dependent protein kinase containing the neural form of type I regulatory subunit have an increased sensitivity to cyclic nucleotides.
• J Biol Chem. 1990; 265: 19502-6
• Display abstract

• Specific isoforms of the cAMP-dependent protein kinase are preferentially expressed within discrete neuronal regions in mouse brain (Cadd and McKnight (1989) Neuron 3, 71-79) suggesting that these subunits might have different functional properties. We have used recombinant techniques to express and purify the type I regulatory subunits, RI alpha and RI beta, the catalytic subunits C alpha and C beta, and then reconstituted holoenzymes with the various combinations of R and C subunits. The ability of the subunits to form inactive holoenzymes and then to be activated in the presence of cyclic nucleotides was examined. Holoenzymes containing C beta had essentially the same activation properties exhibited by C alpha holoenzymes. However, the presence of the neural form of RI, RI beta, led to formation of a holoenzyme which was activated at a 3-7-fold lower concentration of cyclic nucleotides compared to holoenzymes containing RI alpha. Expression of the RI beta protein in discrete regions of the central nervous system may provide a mechanism for increasing the sensitivity of the kinase to what would otherwise be subthreshold levels of stimulation. Two mutant forms of RI beta were constructed that converted the RI beta sequence to that of RI alpha at position 98 (RI beta Ala) or positions 98 and 99 (RI beta Ala/Ile). These sequences form part of a pseudosubstrate site thought to interact with the C subunit. Wild type and mutant R subunits were combined in vitro with purified bovine C subunits and half maximal activation constants (Ka) were determined with cyclic nucleotides. Holoenzymes containing RI beta Ala and RI beta Ala/Ile gave Ka values which were higher than wild type RI beta, with the double mutant shifting toward the Ka value of RI alpha holoenzymes by about 30%. These results suggest that amino acid differences in the pseudosubstrate site may account for some, but not all, of the increased sensitivity to cyclic nucleotides exhibited by RI beta.

• Shabb JB, Ng L, Corbin JD
• One amino acid change produces a high affinity cGMP-binding site in cAMP-dependent protein kinase.
• J Biol Chem. 1990; 265: 16031-4
• Display abstract

• Discrimination between cAMP and cGMP is a critical feature of cAMP- and cGMP-dependent protein kinases. An alanine/threonine difference in the cyclic nucleotide-binding sites has been proposed to provide a structural basis for this functional distinction. Site-directed mutagenesis of this alanine to a threonine in a cAMP-binding site of cAMP kinase produced a mutant with markedly increased cGMP affinity as determined by cGMP binding and protein kinase activation assays. Studies of other mutants at this position support the role of the threonine hydroxyl group as the component that enhances cGMP binding affinity.

• Zhang XP, Ebright RH
• Identification of a contact between arginine-180 of the catabolite gene activator protein (CAP) and base pair 5 of the DNA site in the CAP-DNA complex.
• Proc Natl Acad Sci U S A. 1990; 87: 4717-21
• Display abstract

• We have used site-directed mutagenesis to replace amino acid 1 of the recognition alpha-helix of the catabolite gene activator protein (CAP), Arg-180, with glycine and with alanine. Substitution of Arg-180 of CAP eliminated specificity between G.C, A.T, C.G, and T.A at base pair 5 of the DNA half-site. The effect was position-specific: substitution of Arg-180 did not eliminate specificity between G.C, A.T, C.G, and T.A at base pair 7 of the DNA half-site. We conclude, in agreement with the model for the structure of the CAP-DNA complex [Weber, I. & Steitz, T. (1984) Proc. Natl. Acad. Sci. USA 81, 3973-3977; and Ebright, R., Cossart, P., Gicquel-Sanzey, B. & Beckwith, J. (1984) Proc. Natl. Acad. Sci. USA 81, 7274-7278], that Arg-180 of CAP makes a specificity-determining contact with base pair 5 of the DNA half-site in the CAP-DNA complex. The identification of the contact by Arg-180 in this report, in conjunction with the identification of the contact by Glu-181 in a previous report [Ebright, R., Cossart, P., Gicquel-Sanzey, B. & Beckwith, J. (1984) Nature (London) 311, 232-235], provides information sufficient to define the orientation of the helix-turn-helix motif of CAP with respect to DNA in the CAP-DNA complex.

• Ogreid D, Taylor SS
• Expression and characterization of a truncated form of regulatory subunit of cAMP-dependent protein kinase I lacking binding domain A.
• Adv Second Messenger Phosphoprotein Res. 1990; 24: 196-201

• Ebright RH, Ebright YW, Pendergrast PS, Gunasekera A
• Conversion of a helix-turn-helix motif sequence-specific DNA binding protein into a site-specific DNA cleavage agent.
• Proc Natl Acad Sci U S A. 1990; 87: 2882-6
• Display abstract

• Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein [de Crombrugghe, B., Busby, S. & Buc, H. (1984) Science 224, 831-838; and Pabo, C. & Sauer, R. (1984) Annu. Rev. Biochem. 53, 293-321]. In this work, CAP has been converted into a site-specific DNA cleavage agent by incorporation of the chelator 1,10-phenanthroline at amino acid 10 of the helix-turn-helix motif. [(N-Acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP binds to a 22-base-pair DNA recognition site with Kobs = 1 x 10(8) M-1. In the presence of Cu(II) and reducing agent, [(N-acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP cleaves DNA at four adjacent nucleotides on each DNA strand within the DNA recognition site. The DNA cleavage reaction has been demonstrated using 40-base-pair and 7164-base-pair DNA substrates. The DNA cleavage reaction is not inhibited by dam methylation of the DNA substrate. Such semisynthetic site-specific DNA cleavage agents have potential applications in chromosome mapping, cloning, and sequencing.

• Ebright RH, Gunasekera A, Zhang XP, Kunkel TA, Krakow JS
• Lysine 188 of the catabolite gene activator protein (CAP) plays no role in specificity at base pair 7 of the DNA half site.
• Nucleic Acids Res. 1990; 18: 1457-64
• Display abstract

• Two similar, but not identical, models have been proposed for the amino acid-base pair contacts in the CAP-DNA complex ('model I,' Weber, I. and Steitz, T., Proc. Natl. Acad. Sci. USA, 81, 3973-3977, 1984; 'model II,' Ebright, et al., Proc. Natl. Acad. Sci. USA, 81, 7274-7278, 1984). The most important difference between the two models involves Lys188 of CAP. Model I predicts that Lys188 of CAP makes a specificity determining contact with base pair 7 of the DNA half site. In contrast, model II predicts that Lys188 makes no contact with base pair 7 of the DNA half site. In the present work, we have used site-directed mutagenesis to replace Lys188 of CAP by Asn, an amino acid unable to make the putative contact. We have assessed the specificities of the following proteins, both in vitro and in vivo: wild-type CAP, [Asn188]CAP, [Val181]CAP, and [Val181;Asn188]CAP. The results indicate that Lys188 makes no contribution to specificity at base pair 7 of the DNA half site. We propose, contrary to model I, that Lys188 makes no contact with base pair 7 of the DNA half site.

• Schultz SC, Shields GC, Steitz TA
• Crystallization of Escherichia coli catabolite gene activator protein with its DNA binding site. The use of modular DNA.
• J Mol Biol. 1990; 213: 159-66
• Display abstract

• To obtain crystals of the Escherichia coli catabolite gene activator protein (CAP) complexed with its DNA-binding site, we have searched for crystallization conditions with 26 different DNA segments greater than or equal to 28 base-pairs in length that explore a variety of nucleotide sequences, lengths, and extended 5' or 3' termini. In addition to utilizing uninterrupted asymmetric lac site sequences, we devised a novel approach of synthesizing half-sites that allowed us to efficiently generate symmetric DNA segments with a wide variety of extended termini and lengths in the large size range (greater than or equal to 28 bp) required by this protein. We report three crystal forms that are suitable for X-ray analysis, one of which (crystal form III) gives measurable diffraction amplitudes to 3 A resolution. Additives such as calcium, n-octyl-beta-D-glucopyranoside and spermine produce modest improvements in the quality of diffraction from crystal form III. Adequate stabilization of crystal form III is unexpectedly complex, requiring a greater than tenfold reduction in the salt concentration followed by addition of 2-methyl-2,4-pentanediol and then an increase in the concentration of polyethylene glycol.

• Ringheim GE, Taylor SS
• Effects of cAMP-binding site mutations on intradomain cross-communication in the regulatory subunit of cAMP-dependent protein kinase I.
• J Biol Chem. 1990; 265: 19472-8
• Display abstract

• Each protomer of the regulatory subunit dimer of cAMP-dependent protein kinase contains two tandem and homologous cAMP-binding domains, A and B, and cooperative cAMP binding to these two sites promotes holoenzyme dissociation. Several amino acid residues in the type I regulatory subunit, predicted to lie in close proximity to each bound cyclic nucleotide based on affinity labeling and model building, were replaced using recombinant techniques. The mutations included replacement of 1) Glu-200, predicted to hydrogen bond to the 2'-OH of cAMP bound to site A, with Asp, 2) Tyr-371, the site of affinity labeling with 8-N3-cAMP in site B, with Trp, and 3) Phe-247, the position in site A that is homologous to Tyr-371 in site B, with Tyr. Each mutation caused an approximate 2-fold increase in both the Ka(cAMP) and Kd(cAMP); however, the off-rates for cAMP and the characteristic pattern of affinity labeling with 8-N3-cAMP differed markedly for each mutant protein. Furthermore, these mutations affect the cAMP binding properties not only of the site containing the mutation, but of the adjacent nonmutated site as well, thus confirming that extensive cross-communication occurs between the two cAMP-binding domains. Photoaffinity labeling of the native R-subunit results in the covalent modification of two residues, Trp-260 and Tyr-371, by 8-N3-cAMP bound to sites A and B, respectively, with a stoichiometry of 1 mol of 8-N3-cAMP incorporated per mol of R-monomer (Bubis, J., and Taylor, S. S. (1987) Biochemistry 26, 3478-3486). A stoichiometry of 1 mol of 8-N3-cAMP incorporated per R-monomer was observed for each mutant regulatory subunit as well, even when 2 mol of 8-N3-cAMP were bound per R-monomer; however, the major sites of covalent modification were altered as follows: R(Y371/W), Trp-371; R(E200/D), Tyr-371, and R(F247/Y), Tyr-371.

• Sixl F, King RW, Bracken M, Feeney J
• 19F-n.m.r. studies of ligand binding to 5-fluorotryptophan- and 3-fluorotyrosine-containing cyclic AMP receptor protein from Escherichia coli.
• Biochem J. 1990; 266: 545-52
• Display abstract

• Two fluorine-containing analogues of the cyclic AMP receptor protein (CRP) from Escherichia coli were prepared by biosynthetic incorporation of 5-fluorotryptophan (5-F-Trp) and 3-fluorotyrosine (3-F-Tyr). The 19F-n.m.r. spectrum of the [5-F-Trp]CRP showed two signals corresponding to the two tryptophan residues, and that of the [3-F-Tyr]CRP showed six signals (two overlapping) corresponding to the six tyrosine residues: these results are as expected for a symmetrical dimer. A comparison of the 19F-n.m.r. spectra of the CRP analogues in the presence and in the absence of cyclic AMP reveals that the chemical shifts of both tryptophan residues and of two of the six tyrosine residues show differences. Since none of these residues is in direct contact with the bound nucleotide (although Trp-85 is fairly close), these shift changes must arise from induced conformational effects. The 19F-n.m.r. spectra of complexes with cyclic GMP showed chemical-shift perturbations different from those caused by cyclic AMP, indicating that different conformational changes are induced by the binding of cyclic GMP. The 19F-n.m.r. spectrum of the complex of [3-F-Tyr]CRP with tubercidin 3',5'-(cyclic)monophosphate (which can activate transcription) showed essentially the same chemical-shift changes as seen for the cyclic AMP complex, indicating that similar conformational changes have been induced by the nucleotide binding. [3-F-Tyr]CRP in the presence of an equimolar amount of the 20 bp self-complementary DNA oligomer 5'-AATGTGAGTTAACTCACATT-3' and excess cyclic AMP gave an 19F-n.m.r. spectrum that was almost identical with that for the [3-F-Tyr]CRP-cyclic AMP complex, indicating that the binding of DNA does not induce significant conformational changes involving the tyrosine residues. Proteolysis of [3-F-Tyr]CRP with chymotrypsin produced a 31 kDa fragment that is a dimer containing the cyclic AMP-binding domain. This fragment contains five of the six tyrosine residues, and its 19F-n.m.r. chemical shifts were essentially the same as those of the intact protein except for one missing signal (signal F): this signal could be assigned to Tyr-206 and shown to be unperturbed by the binding of cyclic nucleotide to the intact [3-F-Tyr]CRP. The similarity of the 19F-n.m.r. chemical shifts in the alpha-fragment and the intact CRP indicates that the alpha-fragment retains the same structure as found in the intact protein.(ABSTRACT TRUNCATED AT 400 WORDS)

• Woodford TA, Correll LA, McKnight GS, Corbin JD
• Expression and characterization of mutant forms of the type I regulatory subunit of cAMP-dependent protein kinase. The effect of defective cAMP binding on holoenzyme activation.
• J Biol Chem. 1989; 264: 13321-8
• Display abstract

• The mouse wild type and four mutant regulatory type I (RI) subunits were expressed in Escherichia coli and subjected to kinetic analyses. The defective RI subunits had point mutations in either cAMP-binding site A (G200/E), site B (G324/D, R332/H), or in both binding sites. In addition, a truncated form of RI which lacked the entire cAMP-binding site B was generated. All of the mutant RI subunits which bound [3H]cAMP demonstrated more rapid rates of cAMP dissociation compared to the wild type RI subunit. Dissociation profiles showed only a single dissociation component, suggesting that a single nonmutated binding site was functional. The mutant RI subunits associated with purified native catalytic subunit to form chromatographically separable holoenzyme complexes in which catalytic activity was suppressed. Each of these holoenzymes could be activated but showed varying degrees of cAMP responsiveness with apparent Ka values ranging from 40 nM to greater than 5 microM. The extent to which the mutated cAMP-binding sites were defective was also shown by the resistance of the respective holoenzymes to activation by cAMP analogs selective for the mutated binding sites. Kinetic results support the conclusions that 1) Gly-200 of cAMP-binding site A and Gly-324 or Arg-332 of site B are essential to normal conformation and function, 2) activation of type I cAMP-dependent protein kinase requires that only one of the cAMP-binding sites be functional, 3) mutational inactivation of site B (slow exchange) has a much more drastic effect than that of site A on increasing the Ka of the holoenzyme for cAMP, as well as in altering the rate of cAMP dissociation from the remaining site of the free RI subunit. The strong dependence of one cAMP-binding site on the integrity of the other site suggests a tight association between the two sites.

• Vaney MC, Gilliland GL, Harman JG, Peterkofsky A, Weber IT
• Crystal structure of a cAMP-independent form of catabolite gene activator protein with adenosine substituted in one of two cAMP-binding sites.
• Biochemistry. 1989; 28: 4568-74
• Display abstract

• Catabolite gene activator protein (CAP) in the presence of cAMP stimulates transcription from several operons in Escherichia coli. A cAMP-independent variant, in which Ala-144 is replaced by Thr (CAP91), is activated by analogues of cAMP, such as adenosine, which do not activate the wild-type CAP. In order to test the effect of adenosine on the structure, a crystal of CAP91 grown as a complex with cAMP was soaked in a solution of 10 mM adenosine, and X-ray diffraction data were measured to 3.5-A resolution. The difference Fourier map calculated with phases from the CAP91 structure showed significant negative density at the position of the phosphate of cAMP bound in one subunit of the CAP91 dimer. Adenosine was preferentially substituted for cAMP in the subunit in the "closed" conformation, while the cAMP-binding site of the "open" subunit was apparently still occupied by cAMP. The structure was refined by restrained least-squares methods to an R factor of 20.2%. Adenosine is not bound in exactly the same position as cAMP; instead, the 5'-OH of adenosine is in a new position that allows formation of two hydrogen bonds with Ser-83, replacing two of the three interactions of the phosphate of cAMP with Arg-82 and Ser-83.

• Correll LA, Woodford TA, Corbin JD, Mellon PL, McKnight GS
• Functional characterization of cAMP-binding mutations in type I protein kinase.
• J Biol Chem. 1989; 264: 16672-8
• Display abstract

• A mutant form of the type I regulatory subunit (RI) of cAMP-dependent protein kinase has been cloned and sequenced (Clegg, C. H., Correll, L. A., Cadd, G. C., and McKnight, G. S. (1987) J. Biol. Chem. 262, 13111-13119) which contains two point mutations in the site B cAMP-binding site, a Gly to Asp at position this report, the effect of each independent mutation on the rate of dissociation of cAMP from RI, the cAMP-mediated activation of holoenzyme and the inducibility of cAMP-responsive genes has been characterized. Dissociation of cAMP from either recombinant wild type RI or the B1 mutant demonstrated biphasic kinetics, indicating two sites with different affinities for cAMP. Dissociation from the B2 subunit, however, was monophasic and very rapid indicating that site B had been destroyed and that the rate of dissociation from site A was increased. The cAMP activation constants (Ka) of the wild type and B1 holoenzymes were 40 and 188 nM, respectively, and demonstrated positive cooperativity, with Hill coefficients of 1.61 for the wild type and 1.67 for B1. The B2 holoenzyme required much greater concentrations of cAMP, 4.7 microM, for half-maximal activation and did not display positive cooperativity. Constitutive expression in mouse AtT20 pituitary cells of the B1 mutant resulted in only a small shift in the Ka for kinase activation in these cells compared with B2 expression which increased the Ka by more than 100-fold. Transient expression of the B1 subunit in human JEG-3 choriocarcinoma cells inhibited forskolin activation of a cAMP-responsive promoter by 35% whereas similar expression of the B2 RI subunit inhibited the response by 90%. These results suggest that the Gly to Asp mutation at amino acid 324 completely blocks cAMP binding to site B whereas the Arg to His mutation at position 332 causes a more subtle alteration in cAMP binding. Expression of either mutant RI in animal cells results in a dominant repression of cAMP-dependent protein kinase activity and cAMP-dependent protein kinase-mediated processes.

• Levy FO et al.
• Molecular cloning, complementary deoxyribonucleic acid structure and predicted full-length amino acid sequence of the hormone-inducible regulatory subunit of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase from human testis.
• Mol Endocrinol. 1988; 2: 1364-73
• Display abstract

• In this study, we report the isolation and characterization of a full-length cDNA clone for the hormone-inducible regulatory subunit RII beta (formerly called RII51) of type II cAMP-dependent protein kinase from a human testis cDNA library. The cloned cDNA demonstrated tissue-specific expression of RII beta mRNA in human tissues, with the highest mRNA levels in testis and ovary. The isolated human cDNA clone was 3.3 kilobases (kb) in length and contained 166 base pairs (bp) of G/C-rich 5'-noncoding sequence, an open reading frame of 1254 bp and an A/T-rich 3'-nontranslated region containing 1836 bp followed by an 89 nucleotide long poly(A)-tail. The predicted protein contains 418 amino acids including the start methionine, and the estimated mol wt of human RII beta is 53,856. The nucleotide sequence within the open reading frame and the predicted amino acid sequence of human RII beta are highly conserved compared with partial rat RII beta sequences, displaying 91% and 97% similarity, respectively. Codon preference analysis of the cloned cDNA sequence indicated that the two cAMP-binding domains and the hinge region are highly conserved through evolution, whereas the dimerization domain displayed a codon preference pattern indicative of appearance at a later stage of evolution. The isolated human cDNA detected an FSH- and cAMP-inducible mRNA of 3.2 kb in rat Sertoli cells, thus confirming that the cloned cDNA represents the hormone-inducible regulatory subunit of cAMP-dependent protein kinase. This is the first report documenting the isolation of a full-length cDNA clone for the RII beta of cAMP-dependent protein kinase.

• Ogreid D, Doskeland SO, Gorman KB, Steinberg RA
• Mutations that prevent cyclic nucleotide binding to binding sites A or B of type I cyclic AMP-dependent protein kinase.
• J Biol Chem. 1988; 263: 17397-404
• Display abstract

• Cyclic nucleotide binding and activation properties of cAMP-dependent protein kinases from five independent mutants of S49 mouse lymphoma cells were studied. These mutants were all hemizygous for expression of mutant regulatory (R) subunits of the type I kinase with lesions that altered the electrostatic charge of R subunit: lesions in three of the mutants mapped to cAMP-binding site A, and those in two of the mutants mapped to cAMP-binding site B. A nucleotide mismatch assay using 32P-labeled cRNA and ribonuclease A confirmed and refined localization of the mutations to single amino acid residues implicated in cAMP binding. R subunits from all mutants retained the ability to bind cAMP, but binding behaved as if it were entirely to nonmutated sites: 1) relative affinities of 11 adenine-modified derivatives of cAMP for mutant enzymes were identical to their relative affinities for the site of wild-type kinase that corresponded to the nonmutated site of the mutant; 2) the potencies of these analogs as activators of mutant kinases were strictly correlated with their binding affinities (for wild-type enzyme activation potencies were correlated with mean affinities of the analogs for cAMP-binding sites A and B); 3) combinations of analogs with strong preferences for opposite cAMP-binding sites in wild-type kinase showed no synergism in activating mutant kinases; 4) dissociation of cAMP from mutant kinases was monophasic; and 5) high salt accelerated dissociation of cAMP from kinases with site B lesions but retarded dissociation from those with site A lesions.

• Bubis J, Neitzel JJ, Saraswat LD, Taylor SS
• A point mutation abolishes binding of cAMP to site A in the regulatory subunit of cAMP-dependent protein kinase.
• J Biol Chem. 1988; 263: 9668-73
• Display abstract

• Each regulatory subunit of cAMP-dependent protein kinase has two tandem cAMP-binding sites, A and B, at the carboxyl terminus. Based on sequence homologies with the cAMP-binding domain of the Escherichia coli catabolite gene activator protein, a model has been constructed for each cAMP-binding domain. Two of the conserved features of each cAMP-binding site are an arginine and a glutamic acid which interact with the negatively charged phosphate and with the 2'-OH on the ribose ring, respectively. In the type I regulatory subunit, this arginine in cAMP binding site A is Arg-209. Recombinant DNA techniques have been used to change this arginine to a lysine. The resulting protein binds cAMP with a high affinity and associates with the catalytic subunit to form holoenzyme. The mutant holoenzyme also is activated by cAMP. However, the mutant R-subunit binds only 1 mol of cAMP/R-monomer. Photoaffinity labeling confirmed that the mutant R-subunit has only one functional cAMP-binding site. In contrast to the native R-subunit which is labeled at Trp-260 and Tyr-371 by 8-N3cAMP, the mutant R-subunit is convalently modified at a single site, Tyr-371, which correlates with a functional cAMP-binding site B. The lack of functional cAMP-binding site A also was confirmed by activating the mutant holoenzyme with analogs of cAMP which have a high specificity for either site A or site B. 8-NH2-methyl cAMP which preferentially binds to site B was similar to cAMP in its ability to activate both mutant and wild type holoenzyme whereas N6-monobutyryl cAMP, a site A-specific analog, was a very poor activator of the mutant holoenzyme. The results support the conclusions that 1) Arg-209 is essential for cAMP binding to site A and 2) cAMP binding to domain A is not essential for dissociation of the mutant holoenzyme.

• Bubis J, Saraswat LD, Taylor SS
• Tyrosine-371 contributes to the positive cooperativity between the two cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I.
• Biochemistry. 1988; 27: 1570-6
• Display abstract

• The regulatory (R) subunit of cAMP-dependent protein kinase I has been expressed in Escherichia coli, and oligonucleotide-directed mutagenesis was initiated in order to better understand structural changes that are induced as a consequence of cAMP-binding. Photoaffinity labeling of the type I holoenzyme with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371 [Bubis, J., & Taylor, S.S. (1987) Biochemistry 26, 3478-3486]. The site that was targeted for mutagenesis was Tyr-371. The intention was to establish whether the interactions between the tyrosine ring and the adenine ring of cAMP are primarily hydrophobic in nature or whether the hydroxyl group is critical for cAMP binding and/or for inducing conformational changes. A single base change converted Tyr-371 to Phe. This yielded an R subunit that reassociated with the catalytic subunit to form holoenzyme and bound 2 mol of cAMP/mol of R monomer. The cAMP binding properties of the holoenzyme that was formed with this mutant R subunit, however, were altered: (a) the apparent Kd(cAMP) was shifted from 16 to 60 nM; (b) Scatchard plots showed no cooperativity between the cAMP binding sites in the mutant in contrast to the positive cooperativity that is observed for the wild-type holoenzyme; (c) the Hill coefficient of 1.6 for the wild-type holoenzyme was reduced to 0.99. The Ka's for activation by cAMP were altered in the mutant holoenzyme in a manner that was proportional to the shift in Kd(cAMP).(ABSTRACT TRUNCATED AT 250 WORDS)

• Saraswat LD, Ringheim GE, Bubis J, Taylor SS
• Deletion mutants as probes for localizing regions of subunit interaction in cAMP-dependent protein kinase.
• J Biol Chem. 1988; 263: 18241-6
• Display abstract

• The regulatory subunit of cAMP-dependent protein kinase has a well-defined domain structure, and recombinant DNA techniques have been used to define further the functional properties that are associated with each domain. Our initial question was to define the minimal structural unit that is required for forming a stable complex with the catalytic subunit that will still bind and hence be dissociated by cAMP. To answer these questions, the entire second cAMP-binding domain was deleted using oligonucleotide-directed mutagenesis to introduce a premature stop codon at Trp260. This mutation results in the expression of a stable protein with an Mr of 38,000 based on polyacrylamide gel electrophoresis. The resulting mutant protein is a dimer; and like the native R-subunit, the two protomers of the dimer are cross-linked by disulfide bonds at the amino terminus. The mutant R-subunit binds 1 mol of cAMP/monomer based on equilibrium dialysis. The Kd(cAMP) was 25 nM, which is slightly higher than the Kd(cAMP) for the native R-subunit. The removal of the second cAMP domain does not prevent aggregation with the catalytic subunit, and the inactive holoenzyme complex that is formed in the absence of cAMP can still be dissociated and consequently activated by cAMP. In conjunction with previous results based on limited proteolysis, it is concluded that the region extending from Arg94 to Lys259 constitutes a structural unit that will be sufficient to interact with the catalytic subunit in a cAMP-dependent manner.

• Ringheim GE, Saraswat LD, Bubis J, Taylor SS
• Deletion of cAMP-binding site B in the regulatory subunit of cAMP-dependent protein kinase alters the photoaffinity labeling of site A.
• J Biol Chem. 1988; 263: 18247-52
• Display abstract

• Photoaffinity labeling with 8-azidoadenosine 3':5'-monophosphate is a highly selective method for probing the cAMP-binding sites of the regulatory subunits of cAMP-dependent protein kinase and for identifying specific residues that are in close proximity to the cAMP-binding sites. The cAMP-binding site of a mutant RI-subunit has been characterized here and contrasted to the native RI-subunit. This mutant RI-subunit was generated by oligonucleotide-directed muta-genesis and lacks the entire second cAMP-binding domain which includes both of the residues, Trp260 and Tyr371, that are photolabeled in the native RI-subunit. The mutant RI-subunit, nevertheless, is photoaffinity-labeled with high efficiency, and the residue covalently modified was identified as Tyr244. The position of Tyr244 based on a computer graphic model of cAMP-binding site A is proposed and correlated with the presumed locations of Tyr371 and Trp260 in the native R-subunit. Photoaffinity labeling also can be used to detect functional cAMP-binding sites following electrophoretic transfer of the denatured protein to nitrocellulose. Labeling of the immobilized protein on nitrocellulose required a functional cAMP-binding site A that can be photoaffinity-labeled in solution based on the following criteria. 1) The type I R-subunit is photolabeled, whereas the type II R-subunit is not. A primary feature which distinguishes these two R-subunits is that the RI-subunit is photolabeled at both sites A and B, whereas covalent modification of the RII-subunit occurs only at site B. 2) The truncated mutant of the RI-subunit which lacks the entire second cAMP-binding domain can be photolabeled on nitrocellulose. 3) A mutant RI-subunit which can no longer be photolabeled in site B is still photolabeled on nitrocellulose. 4) A mutation which abolished cAMP binding to site A also abolished photoaffinity labeling after transfer to nitrocellulose.

• Kuno T et al.
• Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase.
• Biochem Biophys Res Commun. 1988; 153: 1244-50
• Display abstract

• The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E. coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites. The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit. When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites. Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.

• Pinkney M, Hoggett JG
• Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.
• Biochem J. 1988; 250: 897-902
• Display abstract

• Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.

• Clegg CH, Cadd GG, McKnight GS
• Genetic characterization of a brain-specific form of the type I regulatory subunit of cAMP-dependent protein kinase.
• Proc Natl Acad Sci U S A. 1988; 85: 3703-7
• Display abstract

• An isoform (RI beta) of the regulatory type I subunit gene of cAMP-dependent protein kinase (EC 2.7.1.37) has been characterized in mouse. The open reading frame of the RI beta cDNA is 72% identical in nucleotide sequence with the previously cloned RI gene, now referred to as RI alpha. Both genes code for a protein of 380 amino acids and their proteins are 82% identical in amino acid sequence. Sequence similarity is highest in the regions that form the pseudosubstrate-binding site of the catalytic subunit and the two cAMP binding domains. The amino-terminal portion shows the greatest dissimilarity, suggesting that the isoforms may differ in their dimerization properties or interaction with other proteins. In contrast to RI alpha, which is constitutively expressed in all tissues, RI beta is expressed in a highly tissue-specific manner. Brain and spinal cord contained significant levels of RI beta mRNA, testis RNA gave a detectable signal, and all other tissues tested were negative. Expression of a RI beta cDNA in NIH 3T3 cells resulted in the appearance of a RI subunit protein that migrated more slowly than RI alpha after NaDodSO4/PAGE. The native form of RI beta in brain could also be distinguished from RI alpha by its abnormal migration on NaDodSO4/PAGE. RI beta protein produced in 3T3 cells was shown to be functional by its ability to form a cAMP-dependent holoenzyme with the catalytic subunit.

• Weber IT
• Crystallizing catabolite gene activator protein with cAMP for structural analysis.
• Methods Enzymol. 1988; 159: 278-85
• Display abstract

• The crystal structure of the CAP dimer with cAMP has provided many insights into the action of this gene regulatory protein. The CAP subunit is divided into two domains that are connected by a hinge region. The carboxy-terminal domains bind to DNA and show both sequence and structural homologies with many other gene regulatory proteins from bacteria and viruses. The amino-terminal domain forms a binding site for cAMP and has been used to model the cAMP-binding domains of the regulatory subunits of mammalian cAMP-dependent protein kinase.

• Ebright RH, Kolb A, Buc H, Kunkel TA, Krakow JS, Beckwith J
• Role of glutamic acid-181 in DNA-sequence recognition by the catabolite gene activator protein (CAP) of Escherichia coli: altered DNA-sequence-recognition properties of [Val181]CAP and [Leu181]CAP.
• Proc Natl Acad Sci U S A. 1987; 84: 6083-7
• Display abstract

• It has been proposed that Glu-181 of the catabolite gene activator protein (CAP) makes direct contact with certain base pairs of the specific DNA site. We have purified wild-type CAP and two substituted CAP variants, [Val181]CAP and [Leu181]CAP, and have assessed the DNA-sequence-recognition properties in vitro with respect to positions 5, 6, 7, 8, and 16 of the DNA site. The data indicate that [Val181]CAP and [Leu181]CAP fail to discriminate between the consensus DNA base pair and the three non-consensus-DNA base pairs at 2-fold-related positions 7 and 16 of the DNA site. In contrast, [Val181]CAP and [Leu181]CAP retain the ability to discriminate between different base pairs at positions 5 and 8 of the DNA site. We conclude that Glu-181 of CAP makes a direct contact with 2-fold-related positions 7 and 16 of the DNA site, as proposed previously based on in vivo results. We propose that upon replacement of Glu-181 by valine or leucine, this contact is eliminated and is replaced by no other functional contact. We estimate that the contact by Glu-181 with each position contributes -0.7 kcal/mol to the total CAP-DNA binding free energy.

• Lacombe ML, Ladant D, Mutzel R, Veron M
• Gene isolation by direct in situ cAMP binding.
• Gene. 1987; 58: 29-36
• Display abstract

• The regulatory subunit of the cAMP-dependent protein kinase expressed in clones isolated by immunoscreening of a lambda gt11 cDNA library from Dictyostelium discoideum exhibits high affinity for cAMP [Mutzel et al., Proc. Natl. Acad. Sci. USA 84 (1987) 6-10]. Based on this property, we have developed a screening procedure to detect in situ cAMP-binding activity directly on phage plaques transferred to nitrocellulose filters. Highly radioactive cAMP was synthesized using [alpha-32P]ATP at 3000 Ci/mmol as the substrate of purified adenylate cyclase from Bordetella pertussis. Filter replicas of the library plated at 3 X 10(4) pfu/dish, were incubated in the presence of 2 nM [32P]cAMP and then washed thoroughly. Three clones out of 1.2 X 10(5) were detected, all of which coded for the regulatory subunit, as judged by hybridization with a specific DNA probe. The cAMP binding to the purified clones was characterized in situ by displacement with specific analogues. The ability to displace labelled cAMP was in accord with the affinities of the analogues previously reported for the regulatory subunit of the Dictyostelium cAMP-dependent protein kinase. We are able to detect fmol levels of regulatory subunit contained in phage plaques and therefore the method could be used to screen libraries from other organisms for proteins exhibiting high affinities for cyclic nucleotides.

• Travers A, Klug A
• Nucleoprotein complexes. DNA wrapping and writhing.
• Nature. 1987; 327: 280-1

• Weber IT, Steitz TA
• Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 A resolution.
• J Mol Biol. 1987; 198: 311-26
• Display abstract

• The structure of a dimer of the Escherichia coli catabolite gene activator protein has been refined at 2.5 A resolution to a crystallographic R-factor of 20.7% starting with coordinates fitted to the map at 2.9 A resolution. The two subunits are in different conformations and each contains one bound molecule of the allosteric activator, cyclic AMP. The amino-terminal domain is linked to the smaller carboxy-terminal domain by a nine-residue hinge region that exists in different conformations in the two subunits, giving rise to approximately a 30 degree rotation between the positions of the small domains relative to the larger domains. The amino-terminal domain contains an antiparallel beta-roll structure in which the interstrand hydrogen bonding is well-determined. The beta-roll can be described as a long antiparallel beta-ribbon that folds into a right-handed supercoil and forms part of the cyclic AMP binding site. Each cyclic AMP molecule is in an anti conformation and has ionic and hydrogen bond interactions with both subunits.

• Rossie S, Gordon D, Catterall WA
• Identification of an intracellular domain of the sodium channel having multiple cAMP-dependent phosphorylation sites.
• J Biol Chem. 1987; 262: 17530-5
• Display abstract

• Cyclic AMP-dependent protein kinase catalyzes the incorporation of 3-4 mol of phosphate into the alpha subunit of rat brain sodium channels in vitro or in situ. Digestion of phosphorylated sodium channels with CNBr yielded three major phosphorylated fragments of 25, 31, and 33 kDa. These fragments were specifically immunoprecipitated with site-directed antisera establishing their location within an intracellular loop between the first and second homologous domains containing residues 448 to 630 of sodium channel RI or residues 450-639 of sodium channel RII. Five of the seven major tryptic phosphopeptides generated from intact sodium channel alpha subunits were contained in each of the 25-, 31-, and 33-kDa CNBr fragments, indicating that most cAMP-dependent phosphorylation sites are in this domain. Since CNBr digestion of sodium channels which had been metabolically labeled with 32P in intact neurons yielded the same phosphorylated fragments, the phosphorylated region we have identified is the major location of phosphorylation in situ. Only serine residues were phosphorylated by cAMP-dependent protein kinase in vitro, while approximately 16% of the phosphorylation in intact neurons was on threonine residues that must lie outside the domain we have identified. Since this domain is phosphorylated in intact neurons, our results show that it is located on the intracellular side of the plasma membrane. These results are considered with respect to models for the transmembrane orientation of the alpha subunit.

• Irwin N, Ptashne M
• Mutants of the catabolite activator protein of Escherichia coli that are specifically deficient in the gene-activation function.
• Proc Natl Acad Sci U S A. 1987; 84: 8315-9
• Display abstract

• In the presence of cyclic AMP, the catabolite activator protein (CAP) of Escherichia coli binds DNA and stimulates transcription at a number of promoters. We have examined a model of CAP bound at the gal promoter and, using directed mutagenesis, have isolated CAP mutants that are analogous to the lambda repressor positive control (pc) mutants. These CAP mutants bind DNA but are defective in stimulating transcription at the gal P1 promoter. These mutants are also altered in positive control at the lac and malT promoters, where CAP binds to sites further upstream from the transcription start site.

• Warwicker J, Engelman BP, Steitz TA
• Electrostatic calculations and model-building suggest that DNA bound to CAP is sharply bent.
• Proteins. 1987; 2: 283-9
• Display abstract

• Two observations suggest that DNA, upon binding to E. coli catabolite gene activator protein (CAP), is sharply bent by a total angle of at least 100-150 degrees: (1) The electrostatic potential field of CAP shows regions of positive potential that form a ramp on 3 sides of the protein. (2) The DNA binding site size as determined by DNA ethylation interference with binding, (Majors: "Control of the E. coli Lac Operon at the Molecular Level." Ph.D. Thesis, Harvard University, Cambridge, 1977) and by relative affinities of DNA fragments of various lengths (Liu-Johnson et al.: Cell 47:995-1005, 1986) requires severe bending of the DNA to maintain its favorable electrostatic contact with the protein.

• Steinberg RA, Russell JL, Murphy CS, Yphantis DA
• Activation of type I cyclic AMP-dependent protein kinases with defective cyclic AMP-binding sites.
• J Biol Chem. 1987; 262: 2664-71
• Display abstract

• Two S49 mouse lymphoma cell variants hemizygous for expression of mutant regulatory (R) subunits of type I cyclic AMP-dependent protein kinase were used to investigate functional consequences of lesions in the putative cAMP-binding sites of R subunit. Kinase activation properties of wild-type and mutant enzymes were compared using cAMP and six site-selective analogs of cAMP. Kinases from both mutant sublines were relatively resistant to cyclic nucleotide-dependent activation, but they were fully activable by at least some effectors. Relative resistances of the mutant kinases varied from about 5-fold for analogs selective for their nonmutated sites to as much as 700-fold for analogs selective for their mutated sites; resistance to cAMP was intermediate. Apparent affinities of wild-type and mutant R subunits for [3H]cAMP were not appreciably different, but competition experiments with site-selective analogs of cAMP suggested that binding of cAMP to mutant R subunits was primarily to their nonmutated sites. Analyses of cooperativity in cyclic nucleotide-dependent activation of mutant kinases, synergism between site I- and site II-selective analogs in activating the mutant enzymes, and dissociation of bound cAMP from mutant R subunits provided additional evidence that the mutations in these strains selectively inactivated single classes of cAMP-binding sites: phenomena attributable in wild-type enzyme to intrachain interactions between sites I and II were always absent or severely diminished in experiments with the mutant enzymes. These results confirm that R subunit sequences implicated in cAMP binding by homology with other cyclic nucleotide-binding proteins actually correspond to functional cAMP-binding sites. Furthermore, occupation of either cAMP-binding site I or II is apparently sufficient for activation of cAMP-dependent protein kinase. The presence of four functional cAMP-binding sites in wild-type kinase enhances the cooperativity and sensitivity of cAMP-mediated activation.

• Weber IT, Gilliland GL, Harman JG, Peterkofsky A
• Crystal structure of a cyclic AMP-independent mutant of catabolite gene activator protein.
• J Biol Chem. 1987; 262: 5630-6
• Display abstract

• Escherichia coli NCR91 synthesizes a mutant form of catabolite gene activator protein (CAP) in which alanine 144 is replaced by threonine. This mutant, which also lacks adenylate cyclase activity, has a CAP phenotype; in the absence of cAMP it is able to express genes that normally require cAMP. CAP91 has been purified and crystallized with cAMP under the same conditions as used to crystallize the wild type CAP X cAMP complex. X-ray diffraction data were measured to 2.4-A resolution and the CAP91 structure was determined using initial model phases from the wild type structure. A difference Fourier map calculated between CAP91 and wild type showed the 2 alanine to threonine sequence changes in the dimer and also a change in orientation of cysteine 178 in one of the subunits. The CAP91 coordinates were refined by restrained least squares to an R factor of 0.186. Differences in the atomic positions of the wild type and mutant protein structures were analyzed by a vector averaging technique. There were small changes that included concerted motions in the small domains, in the hinge between the two domains and in an adjacent loop between beta-strands 4 and 5. The mutation at residue 144 apparently causes changes in the position of some protein atoms that are distal to the mutation site.

• Schlichter D, Miller H, Wicks WD
• On the role of protein kinase subunits in the control of eukaryotic gene expression.
• J Cyclic Nucleotide Protein Phosphor Res. 1986; 11: 149-54
• Display abstract

• Transcriptional regulation by cAMP has been demonstrated for several eukaryotic genes; however, the identity of the protein kinase subunit involved has been a source of debate. Based on homologies with the procaryotic cAMP-binding catabolite activator protein, a recent hypothesis has invoked the regulatory protein RII as the mediator. The evidence currently available on the effects of microinjected kinase subunits suggests, however, that the catalytic subunit is the active factor. Moreover, the proposed homologies between the catabolite activator protein and RII are difficult to reconcile with its proposed mediatory role. We suggest as an alternative hypothesis that a phosphoprotein other than RII may mediate the effects of cAMP on eukaryotic gene expression.

• Angulo J, Krakow JS
• Sensitization of the Escherichia coli cyclic AMP receptor protein to trypsin cleavage by polydeoxyribonucleotides and polyribonucleotides.
• J Biol Chem. 1986; 261: 11315-9
• Display abstract

• In the absence of cAMP the cyclic AMP receptor protein (CRP) is relatively resistant to trypsin whereas the cAMP X CRP complex is attacked yielding N-terminal core fragments of 14,300 and 18,500 Da which still bind cAMP. The DNA X CRP complex formed at low ionic strength in the absence of cAMP is cleaved by trypsin with the formation of 9,700- and 6,000-Da fragments and the concomitant loss of cAMP binding activity. DNA X CRP remains as resistant to attack by subtilisin, clostripain, and the Staphylococcus aureus V8 protease as unliganded CRP but is slowly digested by chymotrypsin. All of the double-stranded polydeoxyribonucleotides and several of the single-stranded polydeoxyribonucleotides and polyribonucleotides tested render CRP sensitive to cleavage by trypsin. CRP is less rapidly cleaved by trypsin in the presence of d(A)n, d(I)n, and r(C)n indicative of a weaker affinity of CRP for these polynucleotides. The 9,700-Da fragment is N-terminal in CRP and probably terminates at Lys-89. The loss of cAMP binding activity following trypsin cleavage of DNA X CRP indicates that regions beyond this residue are important in the function of the cAMP-binding domain of CRP. The 6,000-Da fragment extends from Val-131 to Arg-185 or Lys-188 and contains part of the F helix involved in DNA binding by CRP.

• Buc H
• Mechanism of activation of transcription by the complex formed between cyclic AMP and its receptor in Escherichia coli.
• Biochem Soc Trans. 1986; 14: 196-9

• Connelly PA, Hastings TG, Reimann EM
• Identification of a ternary complex between cAMP and a trimeric form of cAMP-dependent protein kinase.
• J Biol Chem. 1986; 261: 2325-30
• Display abstract

• One of the intermediates involved in dissociation and reassociation of the subunits of the type II cAMP-dependent protein kinase has been characterized. This intermediate can be generated when the protein kinase is prepared from the isolated catalytic subunit (C) and the isolated regulatory subunit-[3H]cAMP complex (R2-[3H]cAMP4) by dialysis for 18 h followed by gel filtration. The intermediate, which could be separated from the holoenzyme and the isolated subunits by polyacrylamide gel electrophoresis, had an apparent molecular weight of 149,000, consistent with an R2C form. Following electrophoresis, measurements of R and bound nucleotide indicated that R2C was half-saturated with [3H]cAMP. The bound [3H]cAMP exhibited biphasic dissociation kinetics indicating that both types of cAMP binding sites were occupied. These findings suggested that the intermediate is R2C-cAMP2. This intermediate was not seen when the dialysis time was increased to 5 days, but could be observed when cAMP was added to the holoenzyme or when holoenzyme was mixed with R2cAMP4 and cAMP. The presence of two occupied cAMP binding sites on this intermediate suggests that there is minimal cooperativity between the two members of the regulatory subunit dimer, i.e. one member of the dimer binds 2 molecules of cAMP while the other binds C.

• Kypr J, Mrazek J
• Possible mechanism of the allosteric activation of cAMP receptor protein.
• Biochem Biophys Res Commun. 1985; 131: 780-5
• Display abstract

• Secondary structure of cAMP receptor protein of E. coli was predicted and compared to its crystal structure in the complex with cAMP solved by McKay and Steitz. The two conformations coincide in the DNA binding domain but strikingly differ in the other domain which binds cAMP and causes protein dimerization. The comparison indicates that cAMP destabilizes a very long helix instead of which sheets are formed creating a hydrophobic pocket where cAMP binds. Consequently, the helix-sheets isomerization and a resulting change in the relative monomer disposition in the dimer appears to be the origin of cAMP-induced allosteric activation of the protein. Extremely long helices were also predicted in the regions of the regulatory subunit of cAMP-dependent protein kinase from bovine cardiac muscle where cAMP binds. It is thus likely that the proposed mechanism of the effect of cAMP on protein structure has wider implications.

• Rannels SR, Cobb CE, Landiss LR, Corbin JD
• The regulatory subunit monomer of cAMP-dependent protein kinase retains the salient kinetic properties of the native dimeric subunit.
• J Biol Chem. 1985; 260: 3423-30
• Display abstract

• Monomeric regulatory subunit (R) fragments of type II cAMP-dependent protein kinase were compared with the parent dimeric R. The monomeric fragments were generated by either endogenous proteolysis of rabbit muscle R or by trypsin treatment of bovine heart R in the holoenzyme form. During isolation of pure R from rabbit muscle, carboxyl-terminal fragments of Mr = 42,000 (42 K) and Mr = 37,000 by denaturing gels are generated by endogenous proteolysis. Although the autophosphorylation site is retained, the 42 K is not dimeric (as is its native 56 K precursor) but, in contrast to the monomeric 37 K product, actively reassociates with purified catalytic subunit (C). Several lines of evidence indicate a type II R origin of the 42 K. N-terminal sequence analysis of the 42 K shows some homology with known bovine RI, RII, and cGMP-dependent protein kinase sequences. Both cyclic nucleotide-binding sites (two/42 K or 37 K) and the site selectivity of cAMP analogs are retained in the monomeric fragments. When purified bovine heart holoenzyme, which contains a dimeric Mr = 56,000 R (denaturing gel analysis) and two C subunits, is treated with trypsin followed by separation procedures, the product is a fully recovered active enzyme with an unaltered ratio of cAMP binding to catalytic activity. From Mr considerations, the product is a dimer containing one intact C and a proteolyzed R of Mr = 48,000 on denaturing gels. This dimeric enzyme is not significantly different from the parent tetramer in cAMP concentration dependence (Hill constant = 1.63), [3H]cAMP dissociation behavior (both intrasubunit cAMP-binding sites are present), stimulation of [3H]cIMP binding by site-selective cAMP analogs, and synergism between two analogs in kinase activation. The data indicate that 1) proteolytic cleavage of the native R dimer can cause monomerization without appreciably affecting the inhibition of C and 2) essentially all of the cAMP binding cooperativity is an intrasubunit interaction.

• Garges S, Adhya S
• Sites of allosteric shift in the structure of the cyclic AMP receptor protein.
• Cell. 1985; 41: 745-51
• Display abstract

• We have characterized crp mutations in E. coli that allow CRP to function without cAMP. crp* mutants carrying a deletion of the gene encoding adenylate cyclase (cya) show significant lac expression. Cyclic GMP, normally an ineffective activator of CRP+, can stimulate these mutant CRP*s to permit greater lac expression in vivo. Cyclic AMP binding to the amino-terminal domain of CRP+ induces an allosteric transition that changes the DNA-binding property of the carboxy domain. The CRP* phenotype is caused by substitution of amino acids with bulkier side chains in the D alpha-helix of the protein's carboxy domain, near the hinge connecting the two domains. These results are consistent with a model in which the mutant CRP*s assume, in part, a conformation normally evoked only by cAMP binding: one in which the relative orientation of the C, D, and F alpha-helices is altered. We define precisely the amino acids of these alpha-helices that interact to cause the allosteric shift.

• Ebright RH, Le Grice SF, Miller JP, Krakow JS
• Analogs of cyclic AMP that elicit the biochemically defined conformational change in catabolite gene activator protein (CAP) but do not stimulate binding to DNA.
• J Mol Biol. 1985; 182: 91-107
• Display abstract

• We have measured the effects on catabolite gene activator protein (CAP) of 22 synthetic analogs of cAMP. Each analog was assayed to test three parameters: (1) binding to CAP; (2) induction of the conformational change in CAP; and (3) activation of transcription. Thus we have identified seven cAMP analogs that bind to CAP as well or better than does cAMP, cause the assayed conformational change in CAP, yet exhibit no ability to activate transcription. We designate these analogs class D. The conformational change elicited in CAP by the class D analogs was further investigated by: (1) sensitivity to the proteolytic enzymes chymotrypsin, Staphylococcus aureus V8 protease, subtilisin and trypsin; (2) formation of inter-subunit covalent crosslinks by 5,5'-dithiobis(2-nitrobenzoic acid); and (3) degree of labeling of cysteine by [3H]N-ethylmaleimide. These experiments failed to detect a conformational difference between the CAP-class D and CAP-cAMP complexes. Filter binding and nuclease protection experiments indicate that the class D analogs do not efficiently support the binding of CAP to DNA. From these results, we suggest that there exists a hitherto undetected event dependent on cAMP, and required for CAP to bind to DNA. We suggest that this event involves a change that takes place in proximity to the N6 atom of cAMP. Three possible interpretations are discussed.

• Bubis J, Taylor SS
• Covalent modification of both cAMP binding sites in cAMP-dependent protein kinase I by 8-azidoadenosine 3',5'-monophosphate.
• Biochemistry. 1985; 24: 2163-70
• Display abstract

• Reconstituted porcine cAMP-dependent protein kinase type I was labeled with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) to study cyclic nucleotide binding and to identify amino acid residues that are either in or in close proximity to the cAMP binding sites. The photoaffinity analogue 8-N3cAMP behaved as cAMP itself with respect to cyclic nucleotide binding. For both cAMP and 8-N3cAMP, 2 mol of nucleotide was bound per mole of type I regulatory subunit monomer (RI), the apparent Kd's observed were approximately 10-17 nM on the basis of either Millipore filtration assays, equilibrium dialysis, or ammonium sulfate precipitation, Scatchard plots showed positive cooperativity, and (4) the Hill coefficients were approximately 1.5-1.6. After photolysis and addition of an excess of cAMP, approximately 1 mol of 8-N3cAMP/mol of RI monomer was covalently incorporated. Tryptic digestion of the labeled protein revealed that two unique tryptic peptides were modified. Proline-271 and tyrosine-371 were identified as the two residues that were covalently modified by 8-N3cAMP in RI. These results contrast with the type II regulatory subunit (RII) where 8-N3cAMP modified covalently a single tyrosine residue [Kerlavage, A. R., & Taylor, S. S. (1980) J. Biol. Chem. 255, 8483-8488]. RI contains two adjacent regions of sequence homology in the COOH-terminal fragment that binds two molecules of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

• Weldon SL, Taylor SS
• Monoclonal antibodies as probes for functional domains in cAMP-dependent protein kinase II.
• J Biol Chem. 1985; 260: 4203-9
• Display abstract

• The antigenic regions of the type II regulatory subunit of cAMP-dependent kinase from bovine heart have been correlated with the previously established domain structure of the molecule. Immunoblotting with both serum and monoclonal antibodies of fragments generated by limited proteolysis or chemical cleavage of the R-subunit established that the major antigenic sites were confined to the amino-terminal portion of the polypeptide chain (residues 1-145). Radioimmunoassays using two different antisera suggested that one or more of the high affinity serum antibody recognition sites were further restricted to residues 91-145. This amino-terminal portion of the R-subunit includes the hinge region which is particularly sensitive to proteolysis, allowing the R-subunit to be cleaved readily into a COOH-terminal domain which retains the cAMP-binding sites and an NH2-terminal fragment which appears to be the major site for interaction of the R-subunits in the native dimer. Monoclonal antibodies that recognized determinants on both sides of this hinge region were characterized and their specific recognition sites localized. Accessibility of antigenic sites in the holoenzyme in contrast to free R2 was compared. Although cAMP did tend to slightly increase the affinity of the holoenzyme for one of the monoclonal antibodies, all of the antigenic sites clearly were exposed and accessible in the holoenzyme. Furthermore, despite the presumed close proximity of antigenic sites to interaction sites between the R- and C-subunits, in no case did binding of antibody to the holoenzyme promote dissociation of the complex. The fact that the monoclonal antibodies would precipitate holoenzyme as well as free R2 was used to ascertain the importance of specific amino acid residues in the interaction of the R- and C-subunits. cAMP-binding domains were isolated following limited proteolysis with chymotrypsin and thermolysin. These fragments differed by only three amino acid residues at the NH2-terminal end. U of these fragments in conjunction with immunoadsorption established that the chymotryptic fragment, which contained the Asp-Arg-Arg preceding the site of autophosphorylation, was capable of forming a stable complex with the C-subunit. In contrast, the thermolytic fragment which differed by only those three residues no longer complexed with the C-subunit, indicating that the arginine residues not only contribute to the specificity of the phosphorylation site but also are an essential component for energetically stabilizing the holoenzyme complex.

• Ebright RH, Cossart P, Gicquel-Sanzey B, Beckwith J
• Molecular basis of DNA sequence recognition by the catabolite gene activator protein: detailed inferences from three mutations that alter DNA sequence specificity.
• Proc Natl Acad Sci U S A. 1984; 81: 7274-8
• Display abstract

• Previously, we reported that substitution of Glu-181 of the catabolite gene activator protein (CAP) by lysine, leucine, or valine results in a protein that has specificity for A X T base pairs at positions 7 and 16 of the DNA recognition site, rather than G X C base pairs as is the case with the wild-type CAP. In this paper, we deduce from these genetic data both (i) the specific chemical interactions by which amino acid side chains at position 181 interact with base pairs 7 and 16 and (ii) the precise alignment between the structures of the CAP and DNA in the intermolecular CAP-DNA complex. Our analysis supports the idea that the two symmetry-related F alpha-helices of the CAP dimer interact with successive major grooves of right-handed B-type DNA [Pabo, C. & Lewis, M. (1982) Nature (London) 298, 443-447; and Steitz, T., Weber, I. & Matthew, J. (1983) Cold Spring Harbor Symp. Quant. Biol. 47, 419-426].

• Weber IT, Steitz TA
• Model of specific complex between catabolite gene activator protein and B-DNA suggested by electrostatic complementarity.
• Proc Natl Acad Sci U S A. 1984; 81: 3973-7
• Display abstract

• Calculation of the electrostatic potential energy surfaces of Escherichia coli catabolite gene activator protein (CAP) dimer suggests a model for the complex between CAP and a specific DNA sequence. The positive electrostatic charge density of CAP lies on the two COOH-terminal domains and about 20-30 A from the molecular 2-fold axis. Assuming that the 2-fold axes of the CAP dimer and the DNA to which it binds are coincident, the positions of the positive electrostatic potential surfaces strongly suggest the rotational orientation of the DNA relative to the protein. A specific complex between CAP and its DNA binding site in the lac operon has been built with the DNA in this orientation. The amino ends of the two protruding F alpha-helices interact in successive major grooves of the DNA. Four side chains emanating from each F helix can form hydrogen bonds with the exposed edges of four bases in the major groove. Electrostatic considerations as well as the necessity to make interactions between CAP and a DNA site as much as 20 base pairs long require us to bend or kink the DNA. In our model of CAP complexed with B-DNA, as with those proposed for Cro and lambda cI repressors, the protruding second helices of the two-helix motif from both subunits interact in successive major grooves of B-DNA. However, unlike Cro and similar to lambda cI, the protruding alpha-helices are nearly parallel to the bases rather than the groove.

• Weber IT, Steitz TA
• A model for the non-specific binding of catabolite gene activator protein to DNA.
• Nucleic Acids Res. 1984; 12: 8475-87
• Display abstract

• The binding of E. coli catabolite gene activator protein (CAP) to non-specific sequences of DNA has been modelled as an electrostatic interaction between four basic side chains of the CAP dimer and the charged phosphates of DNA. Calculation of the electrostatic contribution to the binding free energy at various separations of the two molecules shows that complex formation is favored when CAP and DNA are separated by as much as 12 A. Thus, the long range electrostatic interactions may provide the initial energy for complex formation and also the correct relative orientation of CAP and DNA. The non-specific complex does not involve the penetration of amino acid side chains into the major grooves of DNA and permits 'sliding' of the protein along DNA, which would enhance the rate of association of CAP with the specific site as has been proposed previously for lac repressor. We propose that, as it 'slides', CAP is moving in and out of the major grooves in order to sample the DNA sequence. Recognition of the specific DNA site is achieved by a complementarity in structure and hydrogen bonding between amino acids and the edges of base pairs exposed in the major grooves of DNA.

• de Crombrugghe B, Busby S, Buc H
• Cyclic AMP receptor protein: role in transcription activation.
• Science. 1984; 224: 831-8
• Display abstract

• The structure of this pleiotropic activator of gene transcription in bacteria and its interaction sites at promoter DNA's as well as the role of this protein in the RNA polymerase-promoter interactions are reviewed.

• Steitz TA, Weber IT, Ollis D, Brick P
• Crystallographic studies of protein-nucleic acid interaction: catabolite gene activator protein and the large fragment of DNA polymerase I.
• J Biomol Struct Dyn. 1983; 1: 1023-37
• Display abstract

• Crystals suitable for X-ray crystallographic investigation have been grown of several nucleic acid binding proteins and their analysis is in progress. These include E. coli catabolite gene activator protein (CAP), the large fragment of DNA polymerase I (Pol I fragment), rec A, single strand DNA binding protein, resolvase, lac repressor and lac repressor 'Core', 5S RNA fragment and its complex with L25. Calculation of the electrostatic charge potential of CAP, using coordinates refined at 2.6 A resolution, suggests an orientation for B DNA on this repressor and activator of transcription. Both the electrostatic calculations and detailed model building suggests that the DNA must be bent or kinked on the protein in this orientation in order to make sufficient protein contacts. From a 3.5 A resolution map of Pol I fragment we have been able to obtain a preliminary trace through the polypeptide backbone. The large fragment consists of two domains. The smaller domain binds nucleoside monophosphate at the edge of a mostly parallel beta-pleated sheet, a structure that is reminiscent of kinase and dehydrogenase nucleotide binding domains. The larger domain contains about two thirds of the fragment and is mostly alpha-helical but with at least one four stranded antiparallel beta-sheet. The nucleoside monophosphate binds with its 5' phosphate on the Mg and is apparently in the conformation of nucleotides in B DNA.

• McKay DB, Weber IT, Steitz TA
• Structure of catabolite gene activator protein at 2.9-A resolution. Incorporation of amino acid sequence and interactions with cyclic AMP.
• J Biol Chem. 1982; 257: 9518-24
• Display abstract

• The amino acid sequence of the Escherichia coli catabolite gene activator protein has been fit into a 2.9-A resolution electron density map. Each subunit of the dimer consists of two structurally distinct domains. The larger NH2-terminal domain is seen to bind cyclic AMP and forms all of the contacts between the subunits. The cyclic AMP is completely buried between the interior of the "beta roll" structure of the large domain and a long alpha helix; it makes important hydrogen-bonding interactions with residues from both subunits. The guanidinium group of a buried Arg makes an internal salt link with the phosphate of cyclic AMP. The 6-amino group of adenine interacts simultaneously with both subunits. This interaction with both subunits and the fact that cyclic GMP and cyclic IMP do not activate catabolite gene activator protein suggest that the binding of cyclic AMP may alter the relative orientation of the two subunits, which in turn would change the structure of a DNA binding site that is presumed to span the two smaller domains. The distribution and nature of side chains in the small domain do not rule out the possibility that catabolite gene activator protein binds to left-handed B-DNA.

• Adhya S, Garges S
• How cyclic AMP and its receptor protein act in Escherichia coli.
• Cell. 1982; 29: 287-9

• Garner MM, Revzin A
• Stoichiometry of catabolite activator protein/adenosine cyclic 3',5'-monophosphate interactions at the lac promoter of Escherichia coli.
• Biochemistry. 1982; 21: 6032-6

• Deeley MC, Yanofsky C
• Transcription initiation at the tryptophanase promoter of Escherichia coli K-12.
• J Bacteriol. 1982; 151: 942-51
• Display abstract

• Restriction fragments containing the region preceding the tryptophanase structural gene, tnaA, were used as templates for in vitro transcription experiments. A transcription initiation site was detected that was dependent on the catabolite gene activator protein (CAP) plus cyclic AMP (cAMP). The mRNA produced in vitro was fingerprinted, and the nucleotide at which transcription was initiated was localized to the vicinity of two guanine residues 316 and 318 base pairs upstream of tnaA. A region exhibiting extensive difold symmetry and homology to the CAP binding site adjacent to the lactose operon promoter exists approximately 60 base pairs preceding the site of transcription initiation. Two HinfI restriction sites are located in this region. Restriction enzyme cleavage at these sites was prevented when DNA containing the promoter region was preincubated with CAP and cAMP. RNA polymerase was incapable of protecting these sites against this cleavage. CAP and cAMP addition did not protect against cleavage at a DdeI restriction site located in the -20 region of the promoter. RNA polymerase did protect against DdeI cleavage but only in the presence of CAP and cAMP. Thus, transcription initiation at the tryptophanase promoter involves cAMP-dependent, CAP-facilitated binding of RNA polymerase to the DNA.

• Geahlen RL, Carmichael DF, Hashimoto E, Krebs EG
• Phosphorylation of cAMP-dependent protein kinase subunits.
• Adv Enzyme Regul. 1982; 20: 195-209
• Display abstract

• The cAMP-dependent protein kinases comprise two enzyme forms designated as type I and type II. The type II enzyme can catalyze an autophosphorylation reaction whereby phosphate is transferred from ATP to one seryl residue on each regulatory subunit monomer. Since this reaction can occur in the absence of cAMP-induced enzyme dissociation, it has been used as a probe to identify one site of interaction between the catalytic subunit (C) and the type II regulatory subunit (R11). The type I cAMP-dependent protein kinase does not catalyze an analogous reaction; however, if cGMP-dependent protein kinase is substituted for C, the type I regulatory subunit (R1) becomes phosphorylated. The effects of cyclic nucleotides on this reaction, coupled with the ability of R1 to serve as an inhibitor of cGMP-dependent protein kinase suggest that this phosphorylation also occurs within an important functional domain on R1. A comparison of the autophosphorylation site on R11 with the cGMP-dependent protein kinase catalyzed phosphorylation site on R1 indicates that each modification takes place within a similar proteolytically sensitive region. On each subunit, this sensitive "hinge" region lies distal to the functional domain responsible for regulatory subunit dimerization and proximal to that responsible for cAMP binding. Phosphorylation of the "hinge" region decreases the affinity of each regulatory subunit for C, although the magnitude of this change appears greater for R1 than for R11. Phosphorylation of R1 also reduces the stoichiometry of cAMP binding from two to one mole of cAMP bound per mole of R1 monomer. These results suggest that the "hinge" regions of both R1 and R11 form part of the interaction site between the regulatory subunit and C; and, in the case of R1, it also forms a portion of one of two cAMP-binding sites. The amino acid sequence surrounding the phosphorylated serine of each regulatory subunit has been determined: R11: D-R-R-V-S(P)-V R1: R-R-R-R-G-A-I-S(P)-A It is thought that the number and position of the basic amino acid residues proximal to the modified serine may be responsible, in part, for determining the susceptibility of each site to phosphorylation by cAMP or cGMP-dependent protein kinase. Both R1 and R11 exist as phosphoproteins in vivo. Dephosphorylation of purified "native" phospho-R1 is without effect on the ability of R1 to interact with either C or cAMP. The site phosphorylated in vivo is therefore distinct from that modified in vitro by cGMP-dependent protein kinase. In addition to the autophosphorylation site, R11 possesses a second, less enzymatically reactive, phosphorylation site that is modified in vivo. Dephosphorylation of this site is also without apparent effect on the functional properties of R11. The kinases responsible for catalyzing the phosphorylation of R1 and the cryptic site on R11 and the role that these modifications play in modulating kinase activity are currently unknown but are under active investigation.

• Le Grice SF, Matzura H, Marcoli R, Iida S, Bickle TA
• The catabolite-sensitive promoter for the chloramphenicol acetyl transferase gene is preceded by two binding sites for the catabolite gene activator protein.
• J Bacteriol. 1982; 150: 312-8
• Display abstract

• DNase I protection experiments have indicated that the cyclic AMP-catabolite gene activator protein complex binds to two regions preceding the chloramphenicol acetyl transferase (cat) gene in Escherichia coli. One of these lies adjacent to the RNA polymerase binding site, whereas the second lies approximately 130 base pairs upstream from the starting point of transcription. Additional DNase protection experiments and in vitro transcription experiments with modified templates indicate that the catabolite gene activator protein site proximal to the cat promoter functions independently of the distal site, indicating that in vitro the second of these sites is not required for transcriptional activation of the cat gene.

• Weber IT, Takio K, Titani K, Steitz TA
• The cAMP-binding domains of the regulatory subunit of cAMP-dependent protein kinase and the catabolite gene activator protein are homologous.
• Proc Natl Acad Sci U S A. 1982; 79: 7679-83
• Display abstract

• Comparison of the recently determined amino acid sequences of the regulatory subunit of cAMP-dependent protein kinase (RII) from bovine cardiac muscle and the Escherichia coli catabolite gene activator protein (CAP) shows significant homology. This homology extends over most of the amino-terminal domain in CAP and is particularly good for the region of the beta-roll structure. The RII sequence contains two adjacent and internally homologous regions, both of which have high resemblance to the cAMP-binding domain in CAP. This suggests that the protein kinase regulatory subunit contains two cAMP-binding domains in the carboxyl-terminal region, each having a beta-roll structure similar to that in CAP. The cAMP molecule is expected to bind to the RII within a pocket formed by residues from the beta-roll, as is the case with CAP. One cAMP molecule would interact with residues from about 163 to 220, and the other cAMP would interact with amino acids in the stretch 285-350 of the RII protein kinase sequence. As the carboxyl-terminal domain of CAP shows homologies to the DNA-binding domains of other regulatory proteins, the protein appears to be of modular construction: a DNA-binding domain joined to a cAMP-binding domain.

• Kolb A, Buc H
• Is DNA unwound by the cyclic AMP receptor protein?
• Nucleic Acids Res. 1982; 10: 473-85
• Display abstract

• Superhelical pBR 322 derivatives have been relaxed by eukaryotic topoisomerase I in the presence or in the absence of E. coli cyclic AMP receptor protein (CRP) and of cyclic AMP (cAMP). CRP alone, or cAMP alone do not affect the average linking number of the distribution of the relaxed topoisomers. Hence, they do not unwind the template. In the presence of cAMP, CRP induces a small unwinding. The extent of this unwinding is barely modified when the relaxation is carried out on a similar vector plasmid where the CRP binding site of the lac or of the gal operon has been inserted. Under these conditions, we checked that CRP occupies the lactose control site and that upon addition of RNA polymerase, the corresponding promoter is readily activated. These findings are difficult to reconcile with the proposal that activation of these promoters results from the binding of the CRP-cAMP complex to left-handed DNA sequences.

• Ebright RH, Wong JR
• Mechanism for transcriptional action of cyclic AMP in Escherichia coli: entry into DNA to disrupt DNA secondary structure.
• Proc Natl Acad Sci U S A. 1981; 78: 4011-5
• Display abstract

• Binding analysis with purified bacterial receptor distinguishes two structural domains in cyclic AMP (cAMP). The first, the cyclic phosphate and furanose, constitutes a binding domain. This region is bound tightly to the receptor. The rest of cAMP is not bound; the adenine moiety of cAMP is exposed. Unlike binding, activity of cAMP requires the adenine moiety. To be active, cAMP must have in domain II the base adenine--specifically, its Watson--Crick atoms N-1 and N-6. Analysis of indoleacetic acid, a compound able to replace cAMP at the L-arabinose operon, indicates a similar distinction between binding and active domains. To be active, the indole must have substitution (carboxyl or amide) electronically comparable to the cAMP N-1 and N-6. On this basis, we propose a detailed mechanism for action of cAMP (or indoleacetic acid) in Escherichia coli. We propose that the exposed adenine of cAMP enters into the DNA. The adenine's N-1 and N-6 form hydrogen bonds to a thymine in DNA. This interaction destabilizes the DNA. It enhances transcription. Marked similarities indicate an identical mechanism for the steroid hormones in eukaryotes.

• Garner MM, Revzin A
• Interaction of catabolite activator protein of Escherichia coli with single-stranded deoxyribonucleic acid.
• Biochemistry. 1981; 20: 306-12

• Rannels SR, Corbin JD
• Two different intrachain cAMP binding sites of cAMP-dependent protein kinases.
• J Biol Chem. 1980; 255: 7085-8
• Display abstract

• The regulatory subunits of both isozymes of cAMP-dependent protein kinase bind 2 mol of cAMP/mol of monomer. cAMP dissociation studies indicate similar cAMP binding behavior for each isozyme. Each has two different intrachain cAMP binding components present in approximately equal amounts and the rate of cAMP dissociation is 5- to 10-fold slower from one site (Site 1) than from the other (Site 2). Equilibrium [3H]cAMP binding is inhibited by several competing cyclic nucleotides. Following equilibrium binding using saturating [3H]cAMP in the presence of competing nucleotide, the pattern of release of [3H]cAMP, monitored in the presence of an excess of nonradioactive cAMP, suggests site-specific selectivity of some of the cyclic nucleotides. As compared with cAMP, cIMP prefers Site 2 for both regulatory subunits, whereas N6, O2-dibutyryl-cAMP shows a similar preference only with isozyme II regulatory subunit. 8-Bromo-cAMP, 8-bromo-cGMP, and 8-azido-cAMP prefer Site 1 of both proteins. The results indicate that for each isozyme the two intrachain binding sites have different analogue specificities and cAMP dissociation rates. Site 1 or Site 2 of one isozyme has a similar but not identical cyclic nucleotide specificity and cAMP dissociation rate to the corresponding site of the other isozyme.

• Rannels SR, Corbin JD
• Studies of functional domains of the regulatory subunit from cAMP-dependent protein kinase isozyme I.
• J Cyclic Nucleotide Res. 1980; 6: 201-15
• Display abstract

• Homogenous regulatory subunit from rabbit skeletal muscle cAMP-dependent protein kinase (isozyme I) was partially hydrolyzed with low (1 g/1300 g) or high (1 g/6 g) concentrations of trypsin. After treatment with low trypsin two main peptides (Mr = 35,000 and 12,000) were produced. The cAMP-binding activity (2 mol cAMP/mol of subunit monomer) was recovered in the monomeric Mr = 35,000 peptide. The ability of either fragment to inhibit catalytic subunit activity was lost. Treatment of the regulatory subunit with a high concentration of trypsin yielded three main fragments (Mr = 32,000, 16,000, and 6,000) which could be resolved by Sephadex G-75 and purified further on DEAE-cellulose columns. One of the peptides (Mr = 32,000) bound 2 mol cAMP/mol fragment. The Mr = 16,000 fragment was very labile and bound cAMP with an undetermined stoichiometry. Cyclic AMP dissociation curves for the native regulatory subunit and its Mr = 32,000 component were similar and suggested the presence of two nonidentical binding sites in each monomer. Using the same procedure, the Mr = 16,000 fragment or homogenous cGMP-dependent protein kinase appeared to contain a single type of binding site. Purified Mr = 32,000 fragment was readily converted to the Mr = 16,000 fragment using high trypsin as assessed by protein bands on SDS-disc gels or by following transfer of radioactivity from Mr = 32,000 peptide covalently labeled with 8-N3-[32P] cAMP to radiolabeled Mr = 16,000 fragment. The smallest regulatory subunit fragment (Mr = 6,000) did not bind cAMP, but was dimeric and could be part of the dimerization domain in the native protein. A model is presented to explain the possible structural-functional relationships of the regulatory subunit.

• Rannels SR, Corbin JD
• Characterization of small cAMP-binding fragments of cAMP-dependent protein kinases.
• J Biol Chem. 1979; 254: 8605-10
• Display abstract

• Monomeric cAMP-binding fragments of molecular mass 16,000 and 14,000 daltons were obtained by Sephadex G-75 chromatography of partially trypsin-hydrolyzed regulatory subunits of cAMP-dependent protein kinase isozymes I and II, respectively. The Stokes radii were 19.1 and 16.4 A, the frictional ratios were 1.15 and 1.03, and the sedimentation coefficients were 1.94 and 1.91 S for the 16,000- and 14,000-dalton fragments, respectively. The 16,000-dalton fragment retained specific cyclic nucleotide binding characteristics of the native protein. The specificity of cyclic nucleotide binding to the 14,000-dalton fragment (cAMP greater than cIMP = 8-bromo-cAMP = 8-oxo-cAMP greater than cUMP = cGMP) differed from that of the native subunit (cAMP = 8-oxo-cAMP greater than 8-bromo-cAMP greater than cIMP greater than cUMP = cGMP). The 14,000-dalton fragment bound nearly 1 mol of cAMP/mol of fragment. The binding exchange rate of cAMP was much faster for the 14,000-dalton fragment than for either of the native regulatory subunits or for the 16,000 dalton fragment. Although hemin inhibited cAMP binding to the native regulatory subunits and to the 16,000 dalton fragment, the molecule did not affect cAMP binding to the 14,000-dalton fragment. Both of the native regulatory subunits and the isolated 16,000- and 14,000-dalton fragments could be covalently labeled with the photoaffinity analog, 8-N3-[32P]cAMP. The 14,000-dalton fragment could not be phosphorylated and neither fragment could recombine with the catalytic subunit to inhibit its activity. The results indicate that the functional entities of the regulatory subunit other than cAMP binding are destroyed by trypsin. The properties of the 16,000-dalton fragment suggest that the intact cAMP-binding site is contained in a small trypsin-resistant "core" of the native regulatory subunit. The properties of the 14,000-dalton fragment imply that part of the binding site of the native regulatory subunit was slighlty modified or lost during preparation of this fragment.

• Saxe SA, Revzin A
• Cooperative binding to DNA of catabolite activator protein of Escherichia coli.
• Biochemistry. 1979; 18: 255-63

• Eilen E, Pampeno C, Krakow JS
• Production and properties of the alpha core derived from the cyclic adenosine monophosphate receptor protein of Escherichia coli.
• Biochemistry. 1978; 17: 2469-73

© 2021 EMBL | Privacy Policy