Secondary literature sources for the Ribosomal_S15 domain

For each of the hand selected primary papers associated with the Ribosomal_S15 domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

Crystal structure of the ribosome at 5.5 A resolution.

Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JH, Noller HF
Science. 2001; 292: 883-96

We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.

Several crystal forms of the Bacillus stearothermophilus 50 S ribosomal particles.

Yonath A, Tesche B, Lorenz S, Mussig J, Erdmann VA, Wittmann HG
FEBS Lett. 1983; 154: 15-20

Cooperative interactions of RNA and thiostrepton antibiotic with two domains of ribosomal protein L11.

Xing Y, Draper DE
Biochemistry. 1996; 35: 1581-8

Ribosomal protein L11 interacts with a 58-nucleotide domain of large subunit ribosomal RNA; both the protein and its RNA target have been highly conserved. The antibiotic thiostrepton recognizes the same RNA domain, and binds to the ribosome cooperatively with L11. Experiments presented here show that RNA recognition and thiostrepton cooperativity can be attributed to C- and N-terminal domains of L11, respectively. Under trypsin digestion conditions that degrade Bacillus stearothermophilus L11 to small fragments, the target RNA protects the C-terminal 77 residues from digestion, and thiostrepton and RNA in combination protect the entire protein. A 76-residue C-terminal fragment of L11 was overexpressed and shown to fold into a stable structure binding ribosomal RNA with essentially the same properties as full-length L11. An L11.thiostrepton.RNA complex was 100-200-fold more stable than expected on the basis of L11-RNA and thiostrepton-RNA binding affinities; similar measurements with the C-terminal fragment detected no cooperativity with thiostrepton. L11 function is thus more complex than simple interaction with ribosomal RNA; we suggest that thiostrepton mimics some ribosomal component or factor that normally interacts with the L11 N-terminal domain.

Structure and dynamics of ribosomal RNA.

Woodson SA, Leontis NB
Curr Opin Struct Biol. 1998; 8: 294-300

Over the past two years, progress in X-ray crystallography, NMR spectroscopy and electron microscopy has begun to reveal the complex structure of the RNA within the ribosome. The structures of ribosomal proteins L11 and S15, among others, show how RNA-protein interactions organize the conformation of the junctions between ribosomal RNA helices. Genetic and biochemical methods have also identified a three base-pair switch within the 16S rRNA that is linked to mRNA decoding.

The solution structure of ribosomal protein L18 from Thermus thermophilus reveals a conserved RNA-binding fold.

Woestenenk EA, Gongadze GM, Shcherbakov DV, Rak AV, Garber MB, Hard T, Berglund H
Biochem J. 2002; 363: 553-61

We have determined the solution structure of ribosomal protein L18 from Thermus thermophilus. L18 is a 12.5 kDa protein of the large subunit of the ribosome and binds to both 5 S and 23 S rRNA. In the uncomplexed state L18 folds to a mixed alpha/beta globular structure with a long disordered N-terminal region. We compared our high-resolution structure with RNA-complexed L18 from Haloarcula marismortui and T. thermophilus to examine RNA-induced as well as species-dependent structural differences. We also identified T. thermophilus S11 as a structural homologue and found that the structures of the RNA-recognition sites are conserved. Important features, for instance a bulge in the RNA-contacting beta-sheet, are conserved in both proteins. We suggest that the L18 fold recognizes a specific RNA motif and that the resulting RNA-protein-recognition module is tolerant to variations in sequence.

Crystal structure of a prokaryotic ribosomal protein.

Wilson KS, Appelt K, Badger J, Tanaka I, White SW
Proc Natl Acad Sci U S A. 1986; 83: 7251-5

The structure of ribosomal protein L30 from Bacillus stearothermophilus has been solved to a resolution of 2.5 A. The molecule is somewhat elongated and contains two helices and a three-stranded, anti-parallel beta-pleated sheet. The protein fold, in which helices pack on the same side of the sheet, generates a simple helix-sheet, two-layered motif. It is possible to distinguish three hydrophobic patches on the molecular surface, and one end has six isolated arginine and lysine residues. It is proposed that these reflect sites of protein-protein and protein-RNA interaction, respectively. The protein fold is very similar to that of the only other known ribosomal protein structure, L7/L12 from Escherichia coli, and, based on this similarity, an attempt is made to align the amino acid sequences of the two proteins.

Proteins of the Bacillus stearothermophilus ribosome. A 5 A structure analysis of protein S5.

White SW, Appelt K, Dijk J, Wilson KS
FEBS Lett. 1983; 163: 73-5

The structure of protein S5 from the small subunit of the Bacillus stearothermophilus ribosome is described to a resolution of 5 A. The molecular boundary is visible and shows the protein to be essentially compact although slightly elongated in one dimension.

Location of the binding region for 23S ribosomal RNA on ribosomal protein L2 from Bacillus stearothermophilus.

Watanabe K, Kimura M
Eur J Biochem. 1985; 153: 299-304

Tryptic and chymotryptic digestions of a complex between ribosomal protein L2 and the 23S RNA of Bacillus stearothermophilus produced two protected fragments (designated TF and CF). These protein fragments remained tightly bound to the RNA and were able to rebind specifically to 23S RNA. Amino acid sequence analysis of the isolated fragments revealed that both fragments TF and CF originated from the central half of the protein L2 (positions 60-206 and 58-201) with molecular masses of 15918 Da and 15690 Da respectively. From these results it is concluded that the protected central half is the primary RNA-binding region of the protein L2. A combination of these and other results suggests that the N and/or C-terminal (but not the central) regions may be involved in the peptidyltransferase activity of the 50S subunit.

The domain of the Bacillus subtilis DEAD-box helicase YxiN that is responsible for specific binding of 23S rRNA has an RNA recognition motif fold.

Wang S, Hu Y, Overgaard MT, Karginov FV, Uhlenbeck OC, McKay DB
RNA. 2006; 12: 959-67

The YxiN protein of Bacillus subtilis is a member of the DbpA subfamily of prokaryotic DEAD-box RNA helicases. Like DbpA, it binds with high affinity and specificity to segments of 23S ribosomal RNA as short as 32 nucleotides (nt) that include hairpin 92. Several experiments have shown that the 76-residue carboxy-terminal domain of YxiN is responsible for the high-affinity RNA binding. The domain has been crystallized and its structure has been solved to 1.7 Angstroms resolution. The structure reveals an RNA recognition motif (RRM) fold that is found in many eukaryotic RNA binding proteins; the RRM fold was not apparent from the amino acid sequence. The domain has two solvent exposed aromatic residues at sites that correspond to the aromatic residues of the ribonucleoprotein (RNP) motifs RNP1 and RNP2 that are essential for RNA binding in many RRMs. However, mutagenesis of these residues (Tyr404 and Tyr447) to alanine has little effect on RNA affinity, suggesting that the YxiN domain binds target RNAs in a manner that differs from the binding mode commonly found in many eukaryotic RRMs.

The crystal structure of ribosomal protein L22 from Thermus thermophilus: insights into the mechanism of erythromycin resistance.

Unge J, berg A, Al-Kharadaghi S, Nikulin A, Nikonov S, Davydova N, Nevskaya N, Garber M, Liljas A
Structure. 1998; 6: 1577-86

BACKGROUND: . The ribosomal protein L22 is one of five proteins necessary for the formation of an early folding intermediate of the 23S rRNA. L22 has been found on the cytoplasmic side of the 50S ribosomal subunit. It can also be labeled by an erythromycin derivative bound close to the peptidyl-transfer center at the interface side of the 50S subunit, and the amino acid sequence of an erythromycin-resistant mutant is known. Knowing the structure of the protein may resolve this apparent conflict regarding the location of L22 on the ribosome. RESULTS: . The structure of Thermus thermophilus L22 was solved using X-ray crystallography. L22 consists of a small alpha+beta domain and a protruding beta hairpin that is 30 A long. A large part of the surface area of the protein has the potential to be involved in interactions with rRNA. A structural similarity to other RNA-binding proteins is found, possibly indicating a common evolutionary origin. CONCLUSIONS: . The extensive surface area of L22 has the characteristics of an RNA-binding protein, consistent with its role in the folding of the 23S rRNA. The erythromycin-resistance conferring mutation is located in the protruding beta hairpin that is postulated to be important in L22-rRNA interactions. This region of the protein might be at the erythromycin-binding site close to the peptidyl transferase center, whereas the opposite end may be exposed to the cytoplasm.

Interaction of ribosomal proteins, S6, S8, S15 and S18 with the central domain of 16 S ribosomal RNA.

Svensson P, Changchien LM, Craven GR, Noller HF
J Mol Biol. 1988; 200: 301-8

We have constructed complexes of ribosomal proteins S8, S15, S8 + S15 and S8 + S15 + S6 + S18 with 16 S ribosomal RNA, and probed the RNA moiety with a set of structure-specific chemical and enzymatic probes. Our results show the following effects of assembly of proteins on the reactivity of specific nucleotides in 16 S rRNA. (1) In agreement with earlier work, S8 protects nucleotides in and around the 588-606/632-651 stem from attack by chemical probes; this is supported by protection in and around these same regions from nucleases. In addition, we observe protection of positions 573-575, 583, 812, 858-861 and 865. Several S8-dependent enhancements of reactivity are found, indicating that assembly of this protein is accompanied by conformational changes in 16 S rRNA. These results imply that protein S8 influences a much larger region of the central domain than was previously suspected. (2) Protein S15 protects nucleotides in the 655-672/734-751 stem, in agreement with previous findings. We also find S15-dependent protection of nucleotides in the 724-730 region. Assembly of S15 causes several enhancements of reactivity, the most striking of which are found at G664, A665, G674, and A718. (3) The effects of proteins S6 and S18 are dependent on the simultaneous presence of both proteins, and on the presence of protein S15. S6 + S18-dependent protections are located in the 673-730 and 777-803 regions. We observed some variability in our results with these proteins, depending on the ratio of protein to RNA used, and in different trials using enzymatic probes, possibly due to the limited solubility of protein S18. Consistently reproducible was protection of nucleotides in the 664-676 and 715-729 regions. Among the latter are three of the nucleotides (G664, G674 and A718) that are strongly enhanced by assembly of protein S15. This result suggests that an S15-induced conformational change involving these nucleotides may play a role in the co-operative assembly of proteins S6 and S18.

Discovery and validation of novel and distinct RNA regulators for ribosomal protein S15 in diverse bacterial phyla.

Slinger BL, Deiorio-Haggar K, Anthony JS, Gilligan MM, Meyer MM
BMC Genomics. 2014; 15: 657-657

BACKGROUND: Autogenous cis-regulators of ribosomal protein synthesis play a critical role in maintaining the stoichiometry of ribosome components. Structured portions within an mRNA transcript typically interact with specific ribosomal proteins to prevent expression of the entire operon, thus balancing levels of ribosomal proteins across transcriptional units. Three distinct RNA structures from different bacterial phyla have demonstrated interactions with S15 to regulate gene expression; however, these RNAs are distributed across a small fraction of bacterial diversity. RESULTS: We used comparative genomics in combination with analysis of existing transcriptomic data to identify three novel putative RNA structures associated with the S15 coding region in microbial genomes. These structures are completely distinct from those previously published and encompass potential regulatory regions including ribosome-binding sites. To validate the biological relevance of our findings, we demonstrate that an example of the Alphaproteobacterial RNA from Rhizobium radiobacter specifically interacts with S15 in vitro, and allows in vivo regulation of gene expression in an E. coli reporter system. In addition, structural probing and nuclease protection assays confirm the predicted secondary structure and indicate nucleotides required for protein interaction. CONCLUSIONS: This work illustrates the importance of integrating comparative genomic and transcriptomic approaches during de novo ncRNA identification and reveals a diversity of distinct natural RNA regulators that support analogous biological functions. Furthermore, this work indicates that many additional uncharacterized RNA regulators likely exist within bacterial genomes and that the plasticity of RNA structure allows unique, and likely independently derived, solutions to the same biological problem.

Do mRNA and rRNA binding sites of E.coli ribosomal protein S15 share common structural determinants?

Serganov A, Ennifar E, Portier C, Ehresmann B, Ehresmann C
J Mol Biol. 2002; 320: 963-78

Escherichia coli ribosomal protein S15 recognizes two RNA targets: a three-way junction in 16S rRNA and a pseudoknot structure on its own mRNA. Binding to mRNA occurs when S15 is expressed in excess over its rRNA target, resulting in an inhibition of translation start. The sole apparent similarity between the rRNA and mRNA targets is the presence of a G-U/G-C motif that contributes only modestly to rRNA binding but is essential for mRNA. To get more information on the structural determinants used by S15 to bind its mRNA target as compared to its rRNA site, we used site-directed mutagenesis, substitution by nucleotide analogs, footprinting experiments on both RNA and protein, and graphic modeling. The size of the mRNA-binding site could be reduced to 45 nucleotides, without loss of affinity. This short RNA preferentially folds into a pseudoknot, the formation of which depends on magnesium concentration and temperature. The size of the loop L2 that bridges the two stems of the pseudoknot through the minor groove could not be reduced below nine nucleotides. Then we showed that the pseudoknot recognizes the same side of S15 as 16S rRNA, although shielding a smaller surface area. It turned out that the G-U/G-C motif is recognized from the minor groove in both cases, and that the G-C pair is recognized in a very similar manner. However, the wobble G-U pair of the mRNA is not directly contacted by S15, as in rRNA, but is most likely involved in building a precise conformation of the RNA, essential for binding. Otherwise, unique specific features are utilized, such as the three-way junction in the case of 16S rRNA and the looped out A(-46) for the mRNA pseudoknot.

Shape and compactness of the isolated ribosomal 16 S RNA and its complexes with ribosomal proteins.

Serdyuk IN, Agalarov SC, Sedelnikova SE, Spirin AS, May RP
J Mol Biol. 1983; 169: 409-25

X-ray scattering, neutron scattering and velocity sedimentation techniques were used for studies of ribosomal 16 S RNA in the isolated state and in different complexes with ribosomal proteins. The neutron scattering curve of the ribosomal 30 S subparticle in 42% 2H2O where the protein component is contrast-matched, was taken as a standard of comparison characterizing the dimensions and shape of the 16 S RNA in situ. The following deductions result from the comparisons. The shape of the isolated 16 S RNA at a sufficient Mg2+ concentration (e.g., in the reconstruction buffer) is similar to that of the 16 S RNA in situ, i.e. in the 30 S particle, but it is somewhat less compact. The 16 S RNA in the complex with protein S4 has a shape and compactness similar to those of the isolated 16 S RNA. The 16 S RNA in the complex with four core proteins, namely S4, S7, S8 and S15, has a shape and compactness similar to those of the isolated 16 S RNA. The six ribosomal proteins S4, S7, S8, S15, S16 and S17 are necessary and sufficient for the 16 S RNA to acquire a compactness similar to that within the 30 S particle. The general conclusion is that the overall specific folding of the 16 S RNA is governed and maintained by its own intramolecular interactions, but the additional folding-up (about one-fourth of the linear size of the whole molecule) or the stabilization of the final compactness requires some ribosomal proteins.

Structural preordering in the N-terminal region of ribosomal protein S4 revealed by heteronuclear NMR spectroscopy.

Sayers EW, Gerstner RB, Draper DE, Torchia DA
Biochemistry. 2000; 39: 13602-13

Protein S4, a component of the 30S subunit of the prokaryotic ribosome, is one of the first proteins to interact with rRNA in the process of ribosome assembly and is known to be involved in the regulation of this process. While the structure of the C-terminal 158 residues of Bacillus stearothermophilus S4 has been solved by both X-ray crystallography and NMR, that of the N-terminal 41 residues is unknown. Evidence suggests that the N-terminus is necessary both for the assembly of functional ribosomes and for full binding to 16S RNA, and so we present NMR data collected on the full-length protein (200 aa). Our data indicate that the addition of the N-terminal residues does not significantly change the structure of the C-terminal 158 residues. The data further indicate that the N-terminus is highly flexible in solution, without discernible secondary structure. Nevertheless, structure calculations based on nuclear Overhauser effect spectroscopic data combined with (15)N relaxation data revealed that two short segments in the N-terminus, S(12)RRL(15) and P(30)YPP(33), adopt transiently ordered states in solution. The major conformation of S(12)RRL(15) appears to orient the arginine side chains outward toward the solvent in a parallel fashion, while that of P(30)YPP(33) forms a nascent turn of a polyproline II helix. These segments contain residues that are highly conserved across many prokaryotic species, and thus they are reasonable candidates respectively for sites of interaction with RNA and other ribosomal proteins within the intact ribosome.

In praise of impurity: 30S ribosomal S15 protein-assisted crystallization of turnip yellow mosaic virus proteinase.

Robin C, Beaurepaire L, Chenon M, Jupin I, Bressanelli S
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012; 68: 486-90

Turnip yellow mosaic virus is an excellent model for eukaryotic positive-stranded RNA virus replication. Correct processing of the replication polyprotein is dependent on the virally encoded cysteine proteinase (PRO) domain. Crystalline needles obtained from highly pure preparations of the recombinant 17.6 kDa PRO did not diffract. In contrast, small hexagonal prisms that were obtained together with the needles under the same conditions but from a poorly purified preparation diffracted to 2 A resolution and allowed structure determination by MIRAS. It turned out that the hexagonal crystals contained stoichiometric amounts of PRO and Escherichia coli 30S ribosomal S15, a 10.1 kDa protein commonly co-purified by immobilized metal-affinity chromatography. The solvent content is nearly 70%, with S15 bridging parallel infinite helices of PRO across large solvent channels. With hindsight, this spurious interaction not only yielded diffraction-quality crystals but would also have allowed structure determination by molecular replacement using S15 as a search model and subsequent automatic rebuilding of the asymmetric unit.

Amicoumacin a inhibits translation by stabilizing mRNA interaction with the ribosome.

Polikanov YS, Osterman IA, Szal T, Tashlitsky VN, Serebryakova MV, Kusochek P, Bulkley D, Malanicheva IA, Efimenko TA, Efremenkova OV, Konevega AL, Shaw KJ, Bogdanov AA, Rodnina MV, Dontsova OA, Mankin AS, Steitz TA, Sergiev PV
Mol Cell. 2014; 56: 531-40

We demonstrate that the antibiotic amicoumacin A (AMI) is a potent inhibitor of protein synthesis. Resistance mutations in helix 24 of the 16S rRNA mapped the AMI binding site to the small ribosomal subunit. The crystal structure of bacterial ribosome in complex with AMI solved at 2.4 A resolution revealed that the antibiotic makes contacts with universally conserved nucleotides of 16S rRNA in the E site and the mRNA backbone. Simultaneous interactions of AMI with 16S rRNA and mRNA and the in vivo experimental evidence suggest that it may inhibit the progression of the ribosome along mRNA. Consistent with this proposal, binding of AMI interferes with translocation in vitro. The inhibitory action of AMI can be partly compensated by mutations in the translation elongation factor G.

Comparative structural analysis of eubacterial 5S rRNA by oxidation of adenines in the N-1 position.

Pieler T, Schreiber A, Erdmann VA
Nucleic Acids Res. 1984; 12: 3115-26

Adenines in free 5S rRNA from Escherichia coli, Bacillus stearothermophilus and Thermus thermophilus have been oxidized at their N-1 position using monoperphthalic acid. The determination of the number of adenine 1-N-oxides was on the basis of UV spectroscopic data of the intact molecule. Identification of the most readily accessible nucleotides by sequencing gel analysis reveals that they are located in conserved positions within loops, exposed hairpin loops and single-base bulge loops. Implications for the structure and function of 5S rRNA will be discussed on the basis of this comparative analysis.

Molecular dissection of the pseudoknot governing the translational regulation of Escherichia coli ribosomal protein S15.

Philippe C, Benard L, Portier C, Westhof E, Ehresmann B, Ehresmann C
Nucleic Acids Res. 1995; 23: 18-28

The ribosomal protein S15 controls its own translation by binding to a mRNA region overlapping the ribosome binding site. That region of the mRNA can fold in two mutually exclusive conformations that are in dynamic equilibrium: a structure with two hairpins and a pseudoknot. A mutational analysis provided evidence for the existence and requirement of the pseudoknot for translational control in vivo and S15 recognition in vitro. In this study, we used chemical probing to analyze the structural consequences of mutations and their effect on the stem-loop/pseudoknot equilibrium. Interactions between S15 and the pseudoknot structure were further investigated by footprinting experiments. These data, combined with computer modelling and the previously published data on S15 binding and in vivo control, provide important clues on pseudoknot formation and S15 recognition. An unexpected result is that the relevant control element, here the pseudoknot form, can exist in a variety of topologically equivalent structures recognizable and shapable by S15. S15 sits on the deep groove of the co-axial stack and makes contacts with both stems, shielding the bridging adenine. The only specific sequence determinants are found in the helix common to the pseudoknot and the hairpin structures.

NMR structure of the ribosomal protein L23 from Thermus thermophilus.

Ohman A, Rak A, Dontsova M, Garber MB, Hard T
J Biomol NMR. 2003; 26: 131-7

The ribosomal protein L23 is a component of the large ribosomal subunit in which it is located close to the peptide exit tunnel. In this position L23 plays a central role both for protein secretion and folding. We have determined the solution structure of L23 from Thermus thermophilus. Uncomplexed L23 consists of a well-ordered part, with four anti-parallel beta-strands and three alpha-helices connected as beta-alpha-beta-alpha-beta-beta-alpha, and a large and flexible loop inserted between the third and fourth beta-strand. The observed topology is distantly related to previously known structures, primarily within the area of RNA biochemistry. A comparison with RNA-complexed crystal structures of L23 from T. thermophilus, Deinococcus radiodurans and Haloarcula marismourtui, shows that the conformation of the well-ordered part is very similar in the uncomplexed and complexed states. However, the flexible loop found in the uncomplexed solution structure forms a rigid extended structure in the complexed crystal structures as it interacts with rRNA and becomes part of the exit tunnel wall. Structural characteristics of importance for the interaction with rRNA and with the ribosomal protein L29, as well as the functional role of L23, are discussed.

Archaeal ribosomal protein L1: the structure provides new insights into RNA binding of the L1 protein family.

Nevskaya N, Tischenko S, Fedorov R, Al-Karadaghi S, Liljas A, Kraft A, Piendl W, Garber M, Nikonov S
Structure. 2000; 8: 363-71

BACKGROUND: L1 is an important primary rRNA-binding protein, as well as a translational repressor that binds mRNA. It was shown that L1 proteins from some bacteria and archaea are functionally interchangeable within the ribosome and in the repression of translation. The crystal structure of bacterial L1 from Thermus thermophilus (TthL1) has previously been determined. RESULTS: We report here the first structure of a ribosomal protein from archaea, L1 from Methanococcus jannaschii (MjaL1). The overall shape of the two-domain molecule differs dramatically from that of its bacterial counterpart (TthL1) because of the different relative orientations of the domains. Two strictly conserved regions of the amino acid sequence, each belonging to one of the domains and positioned close to each other in the interdomain cavity of TthL1, are separated by about 25 A in MjaL1 owing to a significant opening of the structure. These regions are structurally highly conserved and are proposed to be the specific RNA-binding sites. CONCLUSIONS: The unusually high RNA-binding affinity of MjaL1 might be explained by the exposure of its highly conserved regions. The open conformation of MjaL1 is strongly stabilized by nonconserved interdomain interactions and suggests that the closed conformations of L1 (as in TthL1) open upon RNA binding. Comparison of the two L1 protein structures reveals a high conformational variability of this ribosomal protein. Determination of the MjaL1 structure offers an additional variant for fitting the L1 protein into electron-density maps of the 50S ribosomal subunit.

Identification of the substrate binding site in the N-terminal TBP-like domain of RNase H3.

Miyashita S, Tadokoro T, Angkawidjaja C, You DJ, Koga Y, Takano K, Kanaya S
FEBS Lett. 2011; 585: 2313-7

Ribonuclease H3 from Bacillus stearothermophilus (Bst-RNase H3) has the N-terminal TBP-like substrate-binding domain. To identify the substrate binding site in this domain, the mutant proteins of the intact protein and isolated N-domain, in which six of the seventeen residues corresponding to those involved in DNA binding of TBP are individually mutated to Ala, were constructed. All of them exhibited decreased enzymatic activities and/or substrate-binding affinities when compared to those of the parent proteins, suggesting that the N-terminal domain of RNase H3 uses the flat surface of the beta-sheet for substrate binding as TBP to bind DNA. This domain may greatly change conformation upon substrate binding.

The structure of Aquifex aeolicus ribosomal protein S8 reveals a unique subdomain that contributes to an extremely tight association with 16S rRNA.

Menichelli E, Edgcomb SP, Recht MI, Williamson JR
J Mol Biol. 2012; 415: 489-502

The assembly of ribonucleoprotein complexes occurs under a broad range of conditions, but the principles that promote assembly and allow function at high temperature are poorly understood. The ribosomal protein S8 from Aquifex aeolicus (AS8) is unique in that there is a 41-residue insertion in the consensus S8 sequence. In addition, AS8 exhibits an unusually high affinity for the 16S ribosomal RNA, characterized by a picomolar dissociation constant that is approximately 26,000-fold tighter than the equivalent interaction from Escherichia coli. Deletion analysis demonstrated that binding to the minimal site on helix 21 occurred at the same nanomolar affinity found for other bacterial species. The additional affinity required the presence of a three-helix junction between helices 20, 21, and 22. The crystal structure of AS8 was solved, revealing the helix-loop-helix geometry of the unique AS8 insertion region, while the core of the molecule is conserved with known S8 structures. The AS8 structure was modeled onto the structure of the 30S ribosomal subunit from E. coli, suggesting the possibility that the unique subdomain provides additional backbone and side-chain contacts between the protein and an unpaired base within the three-way junction of helices 20, 21, and 22. Point mutations in the protein insertion subdomain resulted in a significantly reduced RNA binding affinity with respect to wild-type AS8. These results indicate that the AS8-specific subdomain provides additional interactions with the three-way junction that contribute to the extremely tight binding to ribosomal RNA.

A systematic study of 50S ribosomal subunit purification enabling robust crystallization.

McLellan TJ, Marr ES, Wondrack LM, Subashi TA, Aeed PA, Han S, Xu Z, Wang IK, Maguire BA
Acta Crystallogr D Biol Crystallogr. 2009; 65: 1270-82

A systematic analysis was undertaken to seek correlations between the integrity, purity and activity of 50S ribosomal subunit preparations from Deinococcus radiodurans and their ability to crystallize. Conditions of fermentation, purification and crystallization were varied in a search for crystals that could reliably supply an industrial X-ray crystallography program for the structure-based design of ribosomal antibiotics. A robust protocol was obtained to routinely obtain crystals that gave diffraction patterns extending to 2.9 A resolution and that were large enough to yield a complete data set from a single crystal. To our knowledge, this is the most systematic study of this challenging area so far undertaken. Ribosome crystallization is a complex multi-factorial problem and although a clear correlation of crystallization with subunit properties was not obtained, the search for key factors that potentiate crystallization has been greatly narrowed and promising areas for further inquiry are suggested.

Structure of the pseudouridine synthase RsuA from Haemophilus influenzae.

Matte A, Louie GV, Sivaraman J, Cygler M, Burley SK
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005; 61: 350-4

The structure of the pseudouridine synthase RsuA from Haemophilus influenza, which catalyzes the conversion of uridine to pseudouridine at a single position within 16S ribosomal RNA, has been determined at 1.59 A resolution and compared with that of Escherichia coli RsuA. The H. influenza enzyme contains an N-terminal S4-like alpha3beta4 domain followed by a catalytic domain, as observed in the structure of E. coli RsuA. Whereas the individual domains of E. coli and H. influenza RsuA are structurally similar, their relative spatial disposition differs greatly between the two structures. The former displays an extended open conformation with no direct contacts between the domains, while the latter is in a closed conformation with a large interface between the two domains. Domain closure presents several basic and polar residues into a putative RNA-binding cleft. It is proposed that this relative repositioning of the S4 and catalytic domains is used to modulate the shape and size of the rRNA-binding site in RsuA and in other pseudouridine synthases possessing S4 domains.

High resolution solution structure of ribosomal protein L11-C76, a helical protein with a flexible loop that becomes structured upon binding to RNA.

Markus MA, Hinck AP, Huang S, Draper DE, Torchia DA
Nat Struct Biol. 1997; 4: 70-7

The structure of the C-terminal RNA recognition domain of ribosomal protein L11 has been solved by heteronuclear three-dimensional nuclear magnetic resonance spectroscopy. Although the structure can be considered high resolution in the core, 15 residues between helix alpha 1 and strand beta 1 form an extended, unstructured loop. 15N transverse relaxation measurements suggest that the loop is moving on a picosecond-to-nanosecond time scale in the free protein but not in the protein bound to RNA. Chemical shifts differences between the free protein and the bound protein suggest that the loop as well as the C-terminal end of helix alpha 3 are involved in RNA binding.

Crystallization of the RNA-binding domain of the transcriptional antiterminator protein SacY from Bacillus subtilis.

Manival X, Aymerich S, Strub MP, Dumas C, Kochoyan M, van Tilbeurgh H
Proteins. 1997; 28: 590-4

SacY is the antiterminator protein involved in the induction by sucrose of the expression of the levansucrase gene (sacB) of Bacillus subtilis. In the presence of sucrose, SacY is activated and prevents premature termination of transcription by binding to a RNA-antiterminator (RAT) sequence partially overlapping with the terminator sequence. SacY consists of a RNA-binding N-terminal domain, SacY(1-55), and a regulatory domain, SacY(56-280), sensitive to the sucrose concentration. SacY(1-55) is in itself capable of binding to the RAT sequence and preventing termination independently of the sucrose concentration. In this paper we describe the overexpression, the purification, and the crystallization of SacY(1-55). We obtained six different crystal forms, some of them diffracting to high resolution (> 1.5 A). Self rotation function calculations indicated the presence of a dimer in the asymmetric unit, which is in agreement with a proposed oligomeric state in solution as observed by high-resolution NMR measurements. The crystallization of some site-directed cysteine mutants opens the way of solving the structure by multiple isomorphous replacement.

Crystal structure of the ribosomal protein S6 from Thermus thermophilus.

Lindahl M, Svensson LA, Liljas A, Sedelnikova SE, Eliseikina IA, Fomenkova NP, Nevskaya N, Nikonov SV, Garber MB, Muranova TA, et al.
EMBO J. 1994; 13: 1249-54

The amino acid sequence and crystal structure of the ribosomal protein S6 from the small ribosomal subunit of Thermus thermophilus have been determined. S6 is a small protein with 101 amino acid residues. The 3D structure, which was determined to 2.0 A resolution, consists of a four-stranded anti-parallel beta-sheet with two alpha-helices packed on one side. Similar folding patterns have been observed for other ribosomal proteins and may suggest an original RNA-interacting motif. Related topologies are also found in several other nucleic acid-interacting proteins and based on the assumption that the structure of the ribosome was established early in the molecular evolution, the possibility that an ancestral RNA-interacting motif in ribosomal proteins is the evolutionary origin for the nucleic acid-interacting domain in large classes of ribonucleic acid binding proteins should be considered.

An exceptionally stable helix from the ribosomal protein L9: implications for protein folding and stability.

Kuhlman B, Yang HY, Boice JA, Fairman R, Raleigh DP
J Mol Biol. 1997; 270: 640-7

The ribosomal protein L9 has an unusual structure comprising two compact globular domains connected by a 34 residue alpha-helix. The middle 17 residues of the helix are exposed to solvent while the first seven pack against and form part of the N-terminal domain, and the last ten form part of the C-terminal domain. Here we report results which show that a peptide corresponding to the central helix of L9 is monomeric in aqueous solution and >85% helical at 1 degrees C and 68(+/-7)% helical at 25 degrees C. This is considerably more helical than any other protein fragment studied to date. Another peptide corresponding to the middle 17 residues of the helix is monomeric and is 41(+/-4)% helical at 1 degrees C. Because the central helix has high intrinsic stability the globular N and C-terminal domains will likely be stabilized by their interactions with the helix. Therefore, the stability of the two terminal domains should not be completely independent because both domains gain stability from a shared structural element, the central helix. Also, the ability of the central helix to form native-like structure in isolation highlights a potential role for the helix in the early stages of the folding process.

Mechanisms of allosteric gene regulation by NMR quantification of microsecond-millisecond protein dynamics.

Kleckner IR, Gollnick P, Foster MP
J Mol Biol. 2012; 415: 372-81

The trp RNA-binding attenuation protein (TRAP) is a paradigmatic allosteric protein that regulates the tryptophan biosynthetic genes associated with the trp operon in bacilli. The ring-shaped 11-mer TRAP is activated for recognition of a specific trp-mRNA target by binding up to 11 tryptophan molecules. To characterize the mechanisms of tryptophan-induced TRAP activation, we have performed methyl relaxation dispersion (MRD) nuclear magnetic resonance (NMR) experiments that probe the time-dependent structure of TRAP in the microsecond-to-millisecond "chemical exchange" time window. We find significant side chain flexibility localized to the RNA and tryptophan binding sites of the apo protein and that these dynamics are dramatically reduced upon ligand binding. Analysis of the MRD NMR data provides insights into the structural nature of transiently populated conformations sampled in solution by apo TRAP. The MRD data are inconsistent with global two-state exchange, indicating that conformational sampling in apo TRAP is asynchronous. These findings imply a temporally heterogeneous population of structures that are incompatible with RNA binding and substantiate the study of TRAP as a paradigm for probing and understanding essential dynamics in allosteric, regulatory proteins.

Crystal structure of a bacterial ribonuclease P RNA.

Kazantsev AV, Krivenko AA, Harrington DJ, Holbrook SR, Adams PD, Pace NR
Proc Natl Acad Sci U S A. 2005; 102: 13392-7

The x-ray crystal structure of a 417-nt ribonuclease P RNA from Bacillus stearothermophilus was solved to 3.3-A resolution. This RNA enzyme is constructed from a number of coaxially stacked helical domains joined together by local and long-range interactions. These helical domains are arranged to form a remarkably flat surface, which is implicated by a wealth of biochemical data in the binding and cleavage of the precursors of transfer RNA substrate. Previous photoaffinity crosslinking data are used to position the substrate on the crystal structure and to identify the chemically active site of the ribozyme. This site is located in a highly conserved core structure formed by intricately interlaced long-range interactions between interhelical sequences.

Solution structure of the phosphorylated sites of ribosomal protein S6 by 1H NMR spectroscopy.

Katahira R, Flotow H, Thomas G, Nosaka AY
Int J Pept Protein Res. 1996; 47: 282-8

An increase in the rate of protein synthesis is found to be accompanied by phosphorylation of the 40S ribosomal protein S6. Treatment of S6 by cyanogen bromide produced three fragments, and one of the fragments of S6, which is a C-terminal portion of S6 (M(r) approximately 4,000), contains all phosphorylation sites of S6. The C-terminal fragment of S6 contains seven serines. S6 kinase phosphorylates S6 specifically, i.e. five serines in the C-terminal of S6 are phosphorylated. The three-dimensional structure of S6 peptide was studied in 50% trifluoroethanol/50% H2O solution by 1H NMR with combined use of distance geometry and restrained molecular dynamics calculations. NMR results indicated that it takes an alpha-helix between Glu5 and Arg21 and a distorted helical structure for the following three residues, but no rigid structure was present from Ser25 through the C-terminus and for the N-terminal region (Lys1-Lys4). The specificity of the phosphorylation of the peptide is discussed from a structural aspect.

Mutational analysis of the thermostable arginine repressor from Bacillus stearothermophilus: dissecting residues involved in DNA binding properties.

Karaivanova IM, Weigel P, Takahashi M, Fort C, Versavaud A, Van Duyne G, Charlier D, Hallet JN, Glansdorff N, Sakanyan V
J Mol Biol. 1999; 291: 843-55

Recently the crystal structure of the DNA-unbound form of the full-length hexameric Bacillus stearothermophilus arginine repressor (ArgR) has been resolved, providing a possible explanation for the mechanism of arginine-mediated repressor-operator DNA recognition. In this study we tested some of these functional predictions by performing site-directed mutagenesis of distinct amino acid residues located in two regions, the N-terminal DNA-binding domain and the C-terminal oligomerization domain of ArgR. A total of 15 mutants were probed for their capacity to repress the expression of the reporter argC - lacZ gene fusion in Escherichia coli cells. Substitutions of highly conserved amino acid residues in the alpha2 and alpha3 helices, located in the winged helix-turn-helix DNA-binding motif, reduced repression. Loss of DNA-binding capacity was confirmed in vitro for the Ser42Pro mutant which showed the most pronounced effect in vivo. In E. coli, the wild-type B. stearothermophilus ArgR molecule behaves as a super-repressor, since recombinant E. coli host cells bearing B. stearothermophilusargR on a multicopy vector did not grow in selective minimal medium devoid of arginine and grew, albeit weakly, when l -arginine was supplied. All mutants affected in the DNA-binding domain lost this super-repressor behaviour. Replacements of conserved leucine residues at positions 87 and/or 94 in the C-terminal domain by other hydrophobic amino acid residues proved neutral or caused either derepression or stronger super-repression. Substitution of Leu87 by phenylalanine was found to increase the DNA-binding affinity and the protein solubility in the context of a double Leu87Phe/Leu94Val mutant. Structural modifications occasioned by the various amino acid substitutions were confirmed by circular dichroism analysis and structure modelling.

Structural features of the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA defined using NMR spectroscopy.

Kalurachchi K, Uma K, Zimmermann RA, Nikonowicz EP
Proc Natl Acad Sci U S A. 1997; 94: 2139-44

Ribosomal protein S8 of Escherichia coli plays a key role in 30S ribosomal subunit assembly through its interaction with 16S rRNA. S8 also participates in the translational regulation of ribosomal protein expression through its interaction with spc operon mRNA. The binding site for protein S8 within the 16S rRNA encompasses nucleotides G588 to G604 and C634 to C651 and is composed of two base paired helical regions that flank a phylogenetically conserved core element containing nine residues. We have investigated the structure of the rRNA binding site for S8 both in the free state and in the presence of protein using NMR spectroscopy. The integrity of the two helical segments has been verified, and the presence of G597 x C643 and A596 x U644 base pairs within the conserved core, predicted from comparative analysis, have been confirmed. In addition, we have identified a base triple within the core that is composed of residues A595 x (A596 x U644). The NMR data suggest that S8-RNA interaction is accomplished without significant changes in the RNA. Nonetheless, S8 binding promotes formation of the U598 x A640 base pair and appears to stabilize the G597 x C643 and A596 x U644 base pairs.

The crystal structure of human endothelin.

Janes RW, Peapus DH, Wallace BA
Nat Struct Biol. 1994; 1: 311-9

The three-dimensional structure of the vasoactive polypeptide endothelin, the most potent vasoconstrictor yet identified, has been determined by X-ray crystallography to 2.18 A resolution. This intermediate-sized structure was solved by molecular replacement techniques using a fragment of an NMR-derived model for initial phasing of the data. However, comparisons of the final X-ray structure with the many diverse models derived from NMR data indicate some important differences, especially in the carboxy-terminal region of the molecule: the entire carboxy terminal tail (residues 16-21) is helical in the crystal structure, but not in any of the NMR structures. This may be a functionally significant difference as this region is crucial for receptor binding and vasoactivity.

Structure and dynamics of the anticodon arm binding domain of Bacillus stearothermophilus Tyrosyl-tRNA synthetase.

Guijarro JI, Pintar A, Prochnicka-Chalufour A, Guez V, Gilquin B, Bedouelle H, Delepierre M
Structure. 2002; 10: 311-7

The structure of a recombinant protein, TyrRS(delta4), corresponding to the anticodon arm binding domain of Bacillus stearothermophilus tyrosyl-tRNA synthetase, has been solved, and its dynamics have been studied by nuclear magnetic resonance (NMR). It is the first structure described for such a domain of a tyrosyl-tRNA synthetase. It consists of a five-stranded beta sheet, packed against two alpha helices on one side and one alpha helix on the other side. A large part of the domain is structurally similar to other functionally unrelated RNA binding proteins. The basic residues known to be essential for tRNA binding and charging are exposed to the solvent on the same face of the molecule. The structure of TyrRS(delta4), together with previous mutagenesis data, allows one to delineate the region of interaction with tRNATyr.

Crystal structure and functional characterization of the human RBM25 PWI domain and its flanking basic region.

Gong D, Yang F, Li F, Qian D, Wu M, Shao Z, Wu M, Wu J, Shi Y
Biochem J. 2013; 450: 85-94

Human RBM25 (RNA-binding motif protein 25) is a novel splicing factor that contains a PWI domain, a newly identified RNA/DNA-binding domain, and regulates Bcl-x pre-mRNA alternative splicing. The flanking basic region has been suggested to serve as a co-operative partner of the PWI domain in the binding of nucleic acids, but the structure of this basic region is unknown. In the present paper, we report the crystal structure of the RBM25 PWI domain and its flanking basic region. The PWI domain is revealed to comprise a conserved four-helix bundle, and the flanking basic region forms two alpha-helices and associates with helix H4 of the PWI domain. These interactions promote directly the formation of an enlarged nucleic-acid-binding platform. Structure-guided mutagenesis reveals a positively charged nucleic-acid-binding surface in the RBM25 PWI domain that is entirely different from that in the SRm160 PWI domain. Furthermore, we show that the promotion of the pro-apoptotic Bcl-xS isoform expression by RBM25 is facilitated by the PWI domain in vivo. Thus the present study suggests that the PWI domain plays an important role in the regulation of Bcl-x pre-mRNA alternative splicing.

5S rRNA binding ribosomal proteins from Thermus thermophilus: identification and some structural properties.

Gongadze G, Kashparov I, Lorenz S, Schroeder W, Erdmann VA, Liljas A, Garber M
FEBS Lett. 1996; 386: 260-2

An unusual acidic ribosomal protein from Thermus thermophilus, TL5, that binds to 5S rRNA specifically and strongly, has been investigated. The N-terminal sequence of TL5 does not reveal any homology with known ribosomal proteins. Two large tryptic fragments of TL5 have been isolated and characterized. 5S rRNA protected TL5 and its unstable N-terminal fragment against trypsin action. The 5S rRNA binding ability of TL5 is probably inherent in its N-terminal part. The other 5S rRNA binding ribosomal protein from T. thermophilus, TL4, has been identified as a homolog of the ribosomal protein L5 from Escherichia coli.

Crystal structure of the bacterial conjugation repressor finO.

Ghetu AF, Gubbins MJ, Frost LS, Glover JN
Nat Struct Biol. 2000; 7: 565-9

The conjugative transfer of F-like plasmids is repressed by FinO, an RNA binding protein. FinO interacts with the F-plasmid encoded traJ mRNA and its antisense RNA, FinP, stabilizing FinP against endonucleolytic degradation and facilitating sense-antisense RNA recognition. Here we present the 2.0 A resolution X-ray crystal structure of FinO, lacking its flexible N-terminal extension. FinO adopts a novel, elongated, largely helical conformation. An N-terminal region, previously shown to contact RNA, forms a positively charged alpha-helix (helix 1) that protrudes 45 A from the central core of FinO. A C-terminal region of FinO that is implicated in RNA interactions also extends out from the central body of the protein, adopting a helical conformation and packing against the base of the N-terminal helix. A highly positively charged patch on the surface of the FinO core may present another RNA binding surface. The results of an in vitro RNA duplexing assay demonstrate that the flexible N-terminal region of FinO plays a key role in FinP-traJ RNA recognition, and supports our proposal that this region and the N-terminus of helix 1 interact with and stabilize paired, complementary RNA loops in a kissing complex.

Structure of ribosomal protein L30 from Thermus thermophilus at 1.9 A resolution: conformational flexibility of the molecule.

Fedorov R, Nevskaya N, Khairullina A, Tishchenko S, Mikhailov A, Garber M, Nikonov S
Acta Crystallogr D Biol Crystallogr. 1999; 55: 1827-33

The crystal structure of ribosomal protein L30 from the extreme thermophilic bacterium Thermus thermophilus has been determined at 1. 9 A resolution. The crystals are trigonal and belong to space group P3(2)21, with unit-cell parameters a = b = 63.5, c = 77.8 A, alpha = beta = 90, gamma = 120 degrees and two molecules per asymmetric unit. The structure was solved by the molecular-replacement method with AMoRe and refined with X-PLOR to an R value of 20.3% and an R(free) of 25.3% in the resolution range 8-1.9 A. Detailed analyses of the structures of the two molecules in the asymmetric unit and comparison of T. thermophilus L30 structure with the structure of homologous L30 from Bacillus stearothermophilus reveal two flexible regions at opposite ends of the rather elongated molecule. Such flexibility could be important for the protein fitting in the ribosome. A comparison with B. stearothermophilus L30 shows a higher number of salt bridges and unbound positively charged residues and an increased accessible hydrophobic area on the surface of T. thermophilus L30. This could contribute to the stability of both the extreme thermophile protein and the ribosome as a whole.

S15.Defense mechanisms and development.

Despland JN, Hanin B
Eur Psychiatry. 2000; 15: 77-79

Multiple-site trimethylation of ribosomal protein L11 by the PrmA methyltransferase.

Demirci H, Gregory ST, Dahlberg AE, Jogl G
Structure. 2008; 16: 1059-66

Ribosomal protein L11 is a universally conserved component of the large subunit, and plays a significant role during initiation, elongation, and termination of protein synthesis. In Escherichia coli, the lysine methyltransferase PrmA trimethylates the N-terminal alpha-amino group and the epsilon-amino groups of Lys3 and Lys39. Here, we report four PrmA-L11 complex structures in different orientations with respect to the PrmA active site. Two structures capture the L11 N-terminal alpha-amino group in the active site in a trimethylated post-catalytic state and in a dimethylated state with bound S-adenosyl-L-homocysteine. Two other structures show L11 in a catalytic orientation to modify Lys39 and in a noncatalytic orientation. The comparison of complex structures in different orientations with a minimal substrate recognition complex shows that the binding mode remains conserved in all L11 orientations, and that substrate orientation is brought about by the unusual interdomain flexibility of PrmA.

RNA recognition by transcriptional antiterminators of the BglG/SacY family: mapping of SacY RNA binding site.

Declerck N, Minh NL, Yang Y, Bloch V, Kochoyan M, Aymerich S
J Mol Biol. 2002; 319: 1035-48

Transcriptional antiterminators of the BglG/SacY family are bacterial regulatory proteins able to prevent the premature arrest of transcription through specific binding to a ribonucleic antiterminator (RAT) sequence. The RNA recognition module of these regulators is made of the 55-amino acid long N-terminal domain which can by itself promote efficient antitermination activity in vivo and RNA binding in vitro. The structure of this domain, which was called CAT for co-antiterminator, has first been determined for SacY from Bacillus subtilis and the putative surface contacting RNA has been defined by NMR footprinting. Here we have performed a genetic mapping of the SacY-CAT RNA binding site by substituting 24 amino acid residues including those previously identified by NMR, the highly conserved residues in the 55 homologous antiterminators recognised in the databases and all the positively charged residues. A total of 57 SacY-CAT variants have been constructed and tested in vivo for their antitermination efficiency. A few of these variants were then purified in order to analyse their RNA binding properties by surface plasmon resonance and to check their structural integrity by NMR. The present study validates and clarifies the RNA interacting surface previously mapped by NMR. The residues that are the most intolerant to substitutions, Asn8, His9, Asn10, Gly25, Gly27, and Phe30, are aligned across the CAT dimer interface and form the core of the RNA binding site. Three highly conserved residues stand outside the interaction surface but are essential for maintaining the CAT dimeric structure (Phe47) or may play an important functional role in the full length protein (Glu20 and Lys32). Interestingly, none of the twelve positively charged residues of SacY-CAT are crucial for the antitermination activity. By replacing three Lys residues and combining the Ala26-->Arg mutation that significantly enhanced the affinity for RNA, we engineered a SacY-CAT variant that should be suitable for NMR study of the complex.

Structure analysis of free and bound states of an RNA aptamer against ribosomal protein S8 from Bacillus anthracis.

Davlieva M, Donarski J, Wang J, Shamoo Y, Nikonowicz EP
Nucleic Acids Res. 2014; 42: 10795-808

Several protein-targeted RNA aptamers have been identified for a variety of applications and although the affinities of numerous protein-aptamer complexes have been determined, the structural details of these complexes have not been widely explored. We examined the structural accommodation of an RNA aptamer that binds bacterial r-protein S8. The core of the primary binding site for S8 on helix 21 of 16S rRNA contains a pair of conserved base triples that mold the sugar-phosphate backbone to S8. The aptamer, which does not contain the conserved sequence motif, is specific for the rRNA binding site of S8. The protein-free RNA aptamer adopts a helical structure with multiple non-canonical base pairs. Surprisingly, binding of S8 leads to a dramatic change in the RNA conformation that restores the signature S8 recognition fold through a novel combination of nucleobase interactions. Nucleotides within the non-canonical core rearrange to create a G-(G-C) triple and a U-(A-U)-U quartet. Although native-like S8-RNA interactions are present in the aptamer-S8 complex, the topology of the aptamer RNA differs from that of the helix 21-S8 complex. This is the first example of an RNA aptamer that adopts substantially different secondary structures in the free and protein-bound states and highlights the remarkable plasticity of RNA secondary structure.

The crystal structure of ribosomal protein L14 reveals an important organizational component of the translational apparatus.

Davies C, White SW, Ramakrishnan V
Structure. 1996; 4: 55-66

BACKGROUND: Detailed structural information on ribosomal proteins has increased our understanding of the structure, function and evolution of the ribosome. L14 is one of the most conserved ribosomal proteins and appears to have a central role in the ribonucleoprotein complex. Studies have indicated that L14 occupies a central location between the peptidyl transferase and GTPase regions of the large ribosomal subunit. RESULTS: The crystal structure of L14 from Bacillus stearothermophilus has been solved using a combination of isomorphous replacement and multiwavelength anomalous dispersion (MAD) methods. The structure comprises a five-stranded beta-barrel, a C-terminal loop region that contains two small alpha-helices, and a beta-ribbon that projects from the beta-barrel. An analysis of the structure and the conserved amino acids reveals three surface patches that probably mediate L14-RNA and L14-protein interactions within the ribosome. CONCLUSIONS: The accepted role of ribosomal proteins is to promote the folding and stabilization of ribosomal RNA. The L14 structure is consistent with this notion, and it suggests that the RNA binds in two sites. One RNA-binding site appears to recognize a distinct region of ribosomal RNA during particle assembly. The second site is smaller and may become occupied during the later compaction of the RNA. The surface hydrophobic patch is a likely site of protein-protein interaction, possibly with L19.

Crystal structure of the multidrug efflux transporter AcrB at 3.1A resolution reveals the N-terminal region with conserved amino acids.

Das D, Xu QS, Lee JY, Ankoudinova I, Huang C, Lou Y, DeGiovanni A, Kim R, Kim SH
J Struct Biol. 2007; 158: 494-502

Crystal structures of the bacterial multidrug transporter AcrB in R32 and C2 space groups showing both symmetric and asymmetric trimeric assemblies, respectively, supplemented with biochemical investigations, have provided most of the structural basis for a molecular level understanding of the protein structure and mechanisms for substrate uptake and translocation carried out by this 114-kDa inner membrane protein. They suggest that AcrB captures ligands primarily from the periplasm. Substrates can also enter the inner cavity of the transporter from the cytoplasm, but the exact mechanism of this remains undefined. Analysis of the amino acid sequences of AcrB and its homologs revealed the presence of conserved residues at the N-terminus including two phenylalanines which may be exposed to the cytoplasm. Any potential role that these conserved residues may play in function has not been addressed by existing biochemical or structural studies. Since phenylalanine residues elsewhere in the protein have been implicated in ligand binding, we explored the structure of this N-terminal region to investigate structural determinants near the cytoplasmic opening that may mediate drug uptake. Our structure of AcrB in R32 space group reveals an N-terminus loop, reducing the diameter of the central opening to approximately 15 A as opposed to the previously reported value of approximately 30 A for crystal structures in this space group with disordered N-terminus. Recent structures of the AcrB in C2 space group have revealed a helical conformation of this N-terminus but have not discussed its possible implications. We present the crystal structure of AcrB that reveals the structure of the N-terminus containing the conserved residues. We hope that the structural information provides a structural basis for others to design further biochemical investigation of the role of this portion of AcrB in mediating cytoplasmic ligand discrimination and uptake.

The conformational landscape of the ribosomal protein S15 and its influence on the protein interaction with 16S RNA.

Crety T, Malliavin TE
Biophys J. 2007; 92: 2647-65

The interaction between the ribosomal protein S15 and its binding sites in the 16S RNA was examined from two points of view. First, the isolated protein S15 was studied by comparing NMR conformer sets, available in the PDB and recalculated using the CNS-ARIA protocol. Molecular dynamics (MD) trajectories were then recorded starting from a conformer of each set. The recalculation of the S15 NMR structure, as well as the recording of MD trajectories, reveals that several orientations of the N-terminal alpha-helix alpha1 with respect to the structure core are populated. MD trajectories of the complex between the ribosomal protein S15 and RNA were also recorded in the presence and absence of Mg(2+) ions. The Mg(2+) ions are hexacoordinated by water and RNA oxygens. The coordination spheres mainly interact with the RNA phosphodiester backbone, reducing the RNA mobility and inducing electrostatic screening. When the Mg(2+) ions are removed, the internal mobility of the RNA and of the protein increases at the interaction interface close to the RNA G-U/G-C motif as a result of a gap between the phosphate groups in the UUCG capping tetraloop and of the disruption of S15-RNA hydrogen bonds in that region. On the other hand, several S15-RNA hydrogen bonds are reinforced, and water bridges appear between the three-way junction region and S15. The network of hydrogen bonds observed in the loop between alpha1 and alpha2 is consequently reorganized. In the absence of Mg(2+), this network has the same pattern as the network observed in the isolated protein, where the helix alpha1 is mobile with respect to the protein core. The presence of Mg(2+) ions may thus play a role in stabilizing the orientation of the helix alpha1 of S15.

Inhibition of a ribosome-inactivating ribonuclease: the crystal structure of the cytotoxic domain of colicin E3 in complex with its immunity protein.

Carr S, Walker D, James R, Kleanthous C, Hemmings AM
Structure. 2000; 8: 949-60

BACKGROUND: The cytotoxicity of most ribonuclease E colicins towards Escherichia coli arises from their ability to specifically cleave between bases 1493 and 1494 of 16S ribosomal RNA. This activity is carried by the C-terminal domain of the colicin, an activity which if left unneutralised would lead to destruction of the producing cell. To combat this the host E. coli cell produces an inhibitor protein, the immunity protein, which forms a complex with the ribonuclease domain effectively suppressing its activity. RESULTS: We have solved the crystal structure of the cytotoxic domain of the ribonuclease colicin E3 in complex with its immunity protein, Im3. The structure of the ribonuclease domain, the first of its class, reveals a highly twisted central beta-sheet elaborated with a short N-terminal helix, the residues of which form a well-packed interface with the immunity protein. CONCLUSIONS: The structure of the ribonuclease domain of colicin E3 is novel and forms an interface with its inhibitor which is significantly different in character to that reported for the DNase colicin complexes with their immunity proteins. The structure also gives insight into the mode of action of this class of enzymatic colicins by allowing the identification of potentially catalytic residues. This in turn reveals that the inhibitor does not bind at the active site but rather at an adjacent site, leaving the catalytic centre exposed in a fashion similar to that observed for the DNase colicins. Thus, E. coli appears to have evolved similar methods for ensuring efficient inhibition of the potentially destructive effects of the two classes of enzymatic colicins.

Crystal structure of the BstDEAD N-terminal domain: a novel DEAD protein from Bacillus stearothermophilus.

Carmel AB, Matthews BW
RNA. 2004; 10: 66-74

Most cellular processes requiring RNA structure rearrangement necessitate the action of Asp-Glu-Ala-Asp (DEAD) proteins. Members of the family, named originally for the conserved DEAD amino acid sequence, are thought to disrupt RNA structure and facilitate its rearrangement by unwinding short stretches of duplex RNA. BstDEAD is a novel 436 amino acid representative of the DEAD protein family from Bacillus stearothermophilus that contains all eight conserved motifs found in DEAD proteins and is homologous with other members of the family. Here, we describe the 1.85 A resolution structure of the N-terminal domain (residues 1-211) of BstDEAD (BstDEAD-NT). Similar to the corresponding domains of related helicases, BstDEAD-NT adopts a parallel alpha/beta structure with RecA-like topology. In general, the conserved motifs superimpose on closely related DEAD proteins and on more distantly related helicases such as RecA. This affirms the current belief that the core helicase domains, responsible for mechanistic activity, are structurally similar in DEAD proteins. In contrast, however, the so-called Walker A P-loop, which binds the beta- and gamma-phosphates of ATP, adopts a rarely seen "closed" conformation that would sterically block ATP binding. The closed conformation may be indicative of a general regulatory feature among DEAD proteins (and RNA helicases) that differs from that used by DNA helicases. BstDEAD also contains a unique extension of approximately 60 residues at the C terminus that is highly basic, suggesting that it might bind nucleic acids and, in so doing, confer specificity to the helicase activity of the core region.

N-alkylated oligoamide alpha-helical proteomimetics.

Campbell F, Plante JP, Edwards TA, Warriner SL, Wilson AJ
Org Biomol Chem. 2010; 8: 2344-51

Generic approaches for the design and synthesis of small molecule inhibitors of protein-protein interactions (PPIs) represent a key objective in modern chemical biology. Within this context, the alpha-helix mediated PPIs have received considerable attention as targets for inhibition using small molecules, foldamers and proteomimetics. This manuscript describes a novel N-alkylated aromatic oligoamide proteomimetic scaffold and its solid-phase synthesis--the first time such an approach has been used for proteomimetics. The utility of these scaffolds as proteomimetics is exemplified through the identification of potent microM inhibitors of the p53-hDM2 helix mediated PPI--a key oncogenic target.

The solution structure of the S1 RNA binding domain: a member of an ancient nucleic acid-binding fold.

Bycroft M, Hubbard TJ, Proctor M, Freund SM, Murzin AG
Cell. 1997; 88: 235-42

The S1 domain, originally identified in ribosomal protein S1, is found in a large number of RNA-associated proteins. The structure of the S1 RNA-binding domain from the E. coli polynucleotide phosphorylase has been determined using NMR methods and consists of a five-stranded antiparallel beta barrel. Conserved residues on one face of the barrel and adjacent loops form the putative RNA-binding site. The structure of the S1 domain is very similar to that of cold shock protein, suggesting that they are both derived from an ancient nucleic acid-binding protein. Enhanced sequence searches reveal hitherto unidentified S1 domains in RNase E, RNase II, NusA, EMB-5, and other proteins.

A computational investigation on the connection between dynamics properties of ribosomal proteins and ribosome assembly.

Burton B, Zimmermann MT, Jernigan RL, Wang Y
PLoS Comput Biol. 2012; 8: 1002530-1002530

Assembly of the ribosome from its protein and RNA constituents has been studied extensively over the past 50 years, and experimental evidence suggests that prokaryotic ribosomal proteins undergo conformational changes during assembly. However, to date, no studies have attempted to elucidate these conformational changes. The present work utilizes computational methods to analyze protein dynamics and to investigate the linkage between dynamics and binding of these proteins during the assembly of the ribosome. Ribosomal proteins are known to be positively charged and we find the percentage of positive residues in r-proteins to be about twice that of the average protein: Lys+Arg is 18.7% for E. coli and 21.2% for T. thermophilus. Also, positive residues constitute a large proportion of RNA contacting residues: 39% for E. coli and 46% for T. thermophilus. This affirms the known importance of charge-charge interactions in the assembly of the ribosome. We studied the dynamics of three primary proteins from E. coli and T. thermophilus 30S subunits that bind early in the assembly (S15, S17, and S20) with atomic molecular dynamic simulations, followed by a study of all r-proteins using elastic network models. Molecular dynamics simulations show that solvent-exposed proteins (S15 and S17) tend to adopt more stable solution conformations than an RNA-embedded protein (S20). We also find protein residues that contact the 16S rRNA are generally more mobile in comparison with the other residues. This is because there is a larger proportion of contacting residues located in flexible loop regions. By the use of elastic network models, which are computationally more efficient, we show that this trend holds for most of the 30S r-proteins.

Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16 S RNA.

Brodersen DE, Clemons WM Jr, Carter AP, Wimberly BT, Ramakrishnan V
J Mol Biol. 2002; 316: 725-68

We present a detailed analysis of the protein structures in the 30 S ribosomal subunit from Thermus thermophilus, and their interactions with 16 S RNA based on a crystal structure at 3.05 A resolution. With 20 different polypeptide chains, the 30 S subunit adds significantly to our data base of RNA structure and protein-RNA interactions. In addition to globular domains, many of the proteins have long, extended regions, either in the termini or in internal loops, which make extensive contact to the RNA component and are involved in stabilizing RNA tertiary structure. Many ribosomal proteins share similar alpha+beta sandwich folds, but we show that the topology of this domain varies considerably, as do the ways in which the proteins interact with RNA. Analysis of the protein-RNA interactions in the context of ribosomal assembly shows that the primary binders are globular proteins that bind at RNA multihelix junctions, whereas proteins with long extensions assemble later. We attempt to correlate the structure with a large body of biochemical and genetic data on the 30 S subunit.

Phasing the 30S ribosomal subunit structure.

Brodersen DE, Clemons WM Jr, Carter AP, Wimberly BT, Ramakrishnan V
Acta Crystallogr D Biol Crystallogr. 2003; 59: 2044-50

The methods involved in determining the 850 kDa structure of the 30S ribosomal subunit from Thermus thermophilus were in many ways identical to those that are generally used in standard protein crystallography. This paper reviews and analyses the methods that can be used in phasing such large structures and shows that the anomalous signal collected from heavy-atom compounds bound to the RNA is both necessary and sufficient for ab initio structure determination at high resolution. In addition, measures to counter problems with non-isomorphism and radiation decay are described.

X-ray crystallography shows that translational initiation factor IF3 consists of two compact alpha/beta domains linked by an alpha-helix.

Biou V, Shu F, Ramakrishnan V
EMBO J. 1995; 14: 4056-64

The structures of the two domains of translational initiation factor IF3 from Bacillus stearothermophilus have been solved by X-ray crystallography using single wavelength anomalous scattering and multiwavelength anomalous diffraction. Each of the two domains has an alpha/beta topology, with an exposed beta-sheet that is reminiscent of several ribosomal and other RNA binding proteins. An alpha-helix that protrudes out from the body of the N-terminal domain towards the C-terminal domain suggests that IF3 consists of two RNA binding domains connected by an alpha-helix and that it may bridge two regions of the ribosome. This represents the first high resolution structural information on a translational initiation factor.

Ribosomal proteins and DNA-binding protein II from the extreme thermophile Bacillus caldolyticus.

Beck A, Dijk J, Reinhardt R
Biol Chem Hoppe Seyler. 1987; 368: 121-30

Crystallographic studies, presently on ribosomal and DNA-binding proteins from the moderate thermophile Bacillus stearothermophilus, can be expected to benefit from the use of even more stable proteins from extreme thermophiles. Bacillus caldolyticus, which is able to grow in the temperature range of 70-80 degrees C, appears to be a suitable candidate. We have compared the two bacilli using two criteria: the two-dimensional gel patterns of ribosomal proteins and the properties of DNA-binding protein II. The latter protein is ubiquitous in the eubacterial kingdom and can be purified in large quantities. B. caldolyticus can be grown at 75 degrees C in continuous culture with a generation time of 45-60 min. The yield of ribosomes compares favorably with that of B. stearothermophilus. The gel patterns of the ribosomal proteins are very similar but several differences, in particular among the 50S proteins, are observed. The N-terminal amino-acid sequence of the DNA-binding protein differs in 3 positions (out of 39) from B. stearothermophilus and the protein shows an increased resistance to thermal denaturation. Tetragonal and monoclinic crystals of DNA-binding protein II have been obtained which are suitable for X-ray studies and the diffraction patterns of the two crystal forms are shown.

Interaction of the Bacillus stearothermophilus ribosomal protein S15 with 16 S rRNA: II. Specificity determinants of RNA-protein recognition.

Batey RT, Williamson JR
J Mol Biol. 1996; 261: 550-67

S15 is a primary ribosomal protein that interacts specifically with a three-way junction in the central domain of 16 S rRNA, whose binding induces a conformational change in the RNA. In the accompanying paper, we demonstrated that S15 binds with high affinity to a 61 nucleotide RNA corresponding to the minimal rRNA binding site. Here, the sequence and structural determinants for the RNA in the Bacillus stearothermophilus S15-rRNA interaction have been probed using site-directed mutagenesis, chemical modification interference, and iodine footprinting of phosphorothioate RNA. Mutations and RNA modifications that interfere with protein binding cluster in two distinct regions, one containing an internal loop and the other containing a three-way junction. The internal loop, defined by two A.G base-pairs and a bulged guanosine, is not important for the specific interaction, however, BS15 interacts with a phylogenetically conserved G.U base-pair above this internal loop. Near the three-way junction in helix 22, a bulged adenosine and two base-pairs adjacent to the junction also provide important determinants for BS15 binding. Chemical modification interference also suggests that four highly phylogenetically conserved nucleotides in the three-way junction may form non-canonical G.G and U.A base-pairs that are required for the BS15-rRNA interaction. Ethylation modification interference suggests that BS15 binding is accompanied by a conformational change in the RNA involving orientation of helices 20 and 22 at an acute angle with respect to one another. Projection of the data provided by mutagenesis, chemical modification interference analysis, and iodine footprinting onto a three-dimensional model illustrates that BS15 is likely to interact with the minor groove along an extended face of helix 22.

SbsB structure and lattice reconstruction unveil Ca2+ triggered S-layer assembly.

Baranova E, Fronzes R, Garcia-Pino A, Van Gerven N, Papapostolou D, Pehau-Arnaudet G, Pardon E, Steyaert J, Howorka S, Remaut H
Nature. 2012; 487: 119-22

S-layers are regular two-dimensional semipermeable protein layers that constitute a major cell-wall component in archaea and many bacteria. The nanoscale repeat structure of the S-layer lattices and their self-assembly from S-layer proteins (SLPs) have sparked interest in their use as patterning and display scaffolds for a range of nano-biotechnological applications. Despite their biological abundance and the technological interest in them, structural information about SLPs is limited to truncated and assembly-negative proteins. Here we report the X-ray structure of the SbsB SLP of Geobacillus stearothermophilus PV72/p2 by the use of nanobody-aided crystallization. SbsB consists of a seven-domain protein, formed by an amino-terminal cell-wall attachment domain and six consecutive immunoglobulin-like domains, that organize into a phi-shaped disk-like monomeric crystallization unit stabilized by interdomain Ca(2+) ion coordination. A Ca(2+)-dependent switch to the condensed SbsB quaternary structure pre-positions intermolecular contact zones and renders the protein competent for S-layer assembly. On the basis of crystal packing, chemical crosslinking data and cryo-electron microscopy projections, we present a model for the molecular organization of this SLP into a porous protein sheet inside the S-layer. The SbsB lattice represents a previously undescribed structural model for protein assemblies and may advance our understanding of SLP physiology and self-assembly, as well as the rational design of engineered higher-order structures for biotechnology.

Solution structure of the N-terminal RNP domain of U1A protein: the role of C-terminal residues in structure stability and RNA binding.

Avis JM, Allain FH, Howe PW, Varani G, Nagai K, Neuhaus D
J Mol Biol. 1996; 257: 398-411

The solution structure of a fragment of the human U1A spliceosomal protein containing residues 2 to 117 (U1A117) determined using multi-dimensional heteronuclear NMR is presented. The C-terminal region of the molecule is considerably more ordered in the free protein than thought previously and its conformation is different from that seen in the crystal structure of the complex with U1 RNA hairpin II. The residues between Asp90 and Lys98 form an alpha-helix that lies across the beta-sheet, with residues IIe93, IIe94 and Met97 making contacts with Leu44, Phe56 and IIe58. This interaction prevents solvent exposure of hydrophobic residues on the surface of the beta-sheet, thereby stabilising the protein. Upon RNA binding, helix C moves away from this position, changing its orientation by 135 degrees to allow Tyr13, Phe56 and Gln54 to stack with bases of the RNA, and also allowing Leu44 to contact the RNA. The new position of helix C in the complex with RNA is stabilised by hydrophobic interactions from IIe93 and IIe94 to IIe58, Leu 41, Val62 and His 10, as well as a hydrogen bond between Ser91 and Thr11. The movement of helix C mainly involves changes in the main-chain torsion angles of Thr89, Asp90 and Ser91, the helix thereby acting as a "lid" over the RNA binding surface.

A molecular dynamics study of the C-terminal fragment of the L7/L12 ribosomal protein. Secondary structure motion in a 150 picosecond trajectory.

Aqvist J, van Gunsteren WF, Leijonmarck M, Tapia O
J Mol Biol. 1985; 183: 461-77

A 150 picosecond molecular dynamics computer simulation of the C-terminal fragment of the L7/L12 ribosomal protein from Escherichia coli is reported. The molecular dynamics results are compared with the available high-resolution X-ray data in terms of atomic positions, distances and positional fluctuations. Good agreement is found between the molecular dynamics results and the X-ray data. The form and parameters of the interaction potential energy function and the procedures for deriving it are discussed. Some current misunderstandings concerning the ways of evaluating the efficiency of molecular dynamics algorithms and of application of bond-length constraints in protein simulations are cleared up. The 150 picosecond trajectory has been scanned in a search for correlated motions within and between secondary structure elements. The beta-strands have diffusional stretching modes, and uncorrelated transversal displacements. The dynamic analysis of alpha-helices shows a variety of features. The atomic fluctuations differ between the helix ends; this effect reflects long time-scale motions. Two alpha-helices, alpha A and alpha C, show diffusive longitudinal stretching modes. The third helix, alpha B, has a correlated asymmetric longitudinal stretching; the N-terminal part dominates this behaviour. Furthermore, alpha B presents a librational motion with respect to the other parts of the molecule with a frequency of approximately 5 cm-1. This motion is coupled to helix stretching. Interestingly, the regions of highly conserved residues contain the most mobile parts of the molecule.

Structure of the S15,S6,S18-rRNA complex: assembly of the 30S ribosome central domain.

Agalarov SC, Sridhar Prasad G, Funke PM, Stout CD, Williamson JR
Science. 2000; 288: 107-13

The crystal structure of a 70-kilodalton ribonucleoprotein complex from the central domain of the Thermus thermophilus 30S ribosomal subunit was solved at 2.6 angstrom resolution. The complex consists of a 104-nucleotide RNA fragment composed of two three-helix junctions that lie at the end of a central helix, and the ribosomal proteins S15, S6, and S18. S15 binds the ribosomal RNA early in the assembly of the 30S ribosomal subunit, stabilizing a conformational reorganization of the two three-helix junctions that creates the RNA fold necessary for subsequent binding of S6 and S18. The structure of the complex demonstrates the central role of S15-induced reorganization of central domain RNA for the subsequent steps of ribosome assembly.