NORMAL GENOMIC

• Caillet J et al.
• The modular structure of Escherichia coli threonyl-tRNA synthetase as bothan enzyme and a regulator of gene expression.
• Mol Microbiol. 2003; 47: 961-74
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• In addition to its role in tRNA aminoacylation, Escherichia colithreonyl-tRNA synthetase is a regulatory protein which binds a site,called the operator, located in the leader of its own mRNA and inhibitstranslational initiation by competing with ribosome binding. This workshows that the two essential steps of regulation, operator recognition andinhibition of ribosome binding, are performed by different domains of theprotein. The catalytic and the C-terminal domain of the protein areinvolved in binding the two anticodon arm-like structures in the operatorwhereas the N-terminal domain of the enzyme is responsible for thecompetition with the ribosome. This is the first demonstration of amodular structure for a translational repressor and is reminiscent of thatof transcriptional regulators. The mimicry between the operator and tRNA,suspected on the basis of previous experiments, is further supported bythe fact that identical regions of the synthetase recognize both theoperator and the tRNA anticodon arm. Based on these results, and recentstructural data, we have constructed a computer-derived molecular modelfor the operator-threonyl-tRNA synthetase complex, which sheds light onseveral essential aspects of the regulatory mechanism.

• Choi H, Otten S, Schneider J, McClain WH
• Genetic perturbations of RNA reveal structure-based recognition inprotein-RNA interaction.
• J Mol Biol. 2002; 324: 573-6
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• Protein-RNA recognition is an essential foundation of cellular processes,yet much remains unknown about these important interactions. Therecognition between aminoacyl-tRNA synthetases and their cognate tRNAsubstrates is highly specific and essential for cell viability, due to thenecessity for accurate translation of the genetic code into proteinsequences. We selected an active tRNA that is highly mutated in therecognition nucleotides of the acceptor stem region in the alanine system.The functional properties of this mutant and its secondary derivativesdemonstrate that recognition cannot be reduced to isolated structuralelements, but rather the amino acid acceptor stem is being recognized as aunit.

• Alexander RW, Schimmel P
• Domain-domain communication in aminoacyl-tRNA synthetases.
• Prog Nucleic Acid Res Mol Biol. 2001; 69: 317-49
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• Aminoacyl-tRNA synthetases are modular proteins, with domains that havedistinct roles in the aminoacylation reaction. The catalytic core isresponsible for aminoacyl adenylate formation and transfer of the aminoacid to the 3' end of the bound transfer RNA (tRNA). Appended and inserteddomains contact portions of the tRNA outside the acceptor site andcontribute to the efficiency and specificity of aminoacylation. Someaminoacyl-tRNA synthetases also have distinct editing activities that arelocalized to unique domains. Efficient aminoacylation and editing requirecommunication between RNA-binding and catalytic domains, and can beconsidered as a signal transduction system. Here, evidence fordomain-domain communication in aminoacyl-tRNA synthetases is summarized,together with insights from structural analysis.

• Cura V, Moras D, Kern D
• Sequence analysis and modular organization of threonyl-tRNA synthetasefrom Thermus thermophilus and its interrelation with threonyl-tRNAsynthetases of other origins.
• Eur J Biochem. 2000; 267: 379-93
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• The gene encoding threonyl-tRNA synthetase (Thr-tRNA synthetase) from theextreme thermophilic eubacterium Thermus thermophilus HB8 has been clonedand sequenced. The ORF encodes a polypeptide chain of 659 amino acids (Mr75 550) that shares strong similarities with other Thr-tRNA synthetases.Comparative analysis with the three-dimensional structure of othersubclass IIa synthetases shows it to be organized into four structuralmodules: two N-terminal modules specific to Thr-tRNA synthetases, acatalytic core and a C-terminal anticodon-binding module. Comparison withthe three-dimensional structure of Escherichia coli Thr-tRNA synthetase incomplex with tRNAThr enabled identification of the residues involved insubstrate binding and catalytic activity. Analysis by atomic absorptionspectrometry of the enzyme overexpressed in E. coli revealed the presencein each monomer of one tightly bound zinc atom, which is essential foractivity. Despite strong similarites in modular organization, Thr-tRNAsynthetases diverge from other subclass IIa synthetases on the basis oftheir N-terminal extensions. The eubacterial and eukaryotic enzymespossess a large extension folded into two structural domains, N1 and N2,that are not significantly similar to the shorter extension of thearchaebacterial enzymes. Investigation of a truncated Thr-tRNA synthetasedemonstrated that domain N1 is not essential for tRNA charging. Thr-tRNAsynthetase from T. thermophilus is of the eubacterial type, in contrast toother synthetases from this organism, which exhibit archaebacterialcharacteristics. Alignments show conservation of part of domain N2 in theC-terminal moiety of Ala-tRNA synthetases. Analysis of the nucleotidesequence upstream from the ORF showed the absence of both anyanticodon-like stem-loop structure and a loop containing sequencescomplementary to the anticodon and the CCA end of tRNAThr. This means thatthe expression of Thr-tRNA synthetase in T. thermophilus is not regulatedby the translational and trancriptional mechanisms described for E. colithrS and Bacillus subtilis thrS and thrZ. Here we discuss our results inthe context of evolution of the threonylation systems and of the positionof T. thermophilus in the phylogenic tree.

• Cusack S, Yaremchuk A, Krikliviy I, Tukalo M
• tRNA(Pro) anticodon recognition by Thermus thermophilus prolyl-tRNAsynthetase.
• Structure. 1998; 6: 101-8
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• BACKGROUND: Most aminoacyl-tRNA synthetases (aaRSs) specifically recognizeall or part of the anticodon triplet of nucleotides of their cognatetRNAs. Class IIa and class IIb aaRSs possess structurally distinct tRNAanticodon-binding domains. The class IIb enzymes (LysRS, AspRS and AsnRS)have an N-terminal beta-barrel domain (OB-fold); the interactions of thisdomain with the anticodon stem-loop are structurally well characterisedfor AspRS and LysRS. Four out of five class IIa enzymes (ProRS, ThrRS,HisRS and GlyRS, but not SerRS) have a C-terminal anticodon-binding domainwith an alpha/beta fold, not yet found in any other protein. The mode ofRNA binding by this domain is hitherto unknown as is the rationale, ifany, behind classification of anticodon-binding domains for differentaaRSs. RESULTS: The crystal structure of Thermus thermophilus prolyl-tRNAsynthetase (ProRSTT) in complex with tRNA(Pro) has been determined at 3.5A resolution by molecular replacement using the native enzyme structure.One tRNA molecule, of which only the lower two-thirds is well ordered, isfound bound to the synthetase dimer. The C-terminal anticodon-bindingdomain binds to the anticodon stem-loop from the major groove side.Binding to tRNA by ProRSTT is reminiscent of the interaction of class IIbenzymes with cognate tRNAs, but only three of the anticodon-loop basesbecome splayed out (bases 35-37) rather than five (bases 33-37) in thecase of class IIb enzymes. The two anticodon bases conserved in alltRNA(Pro), G35 and G36, are specifically recognised by ProRSTT.CONCLUSIONS: For the synthetases possessing the class IIaanticodon-binding domain (ProRS, ThrRS and GlyRS, with the exception ofHisRS), the two anticodon bases 35 and 36 are sufficient to uniquelyidentify the cognate tRNA (GG for proline, GU for threonine, CC forglycine), because these amino acids occupy full codon groups. Thestructure of ProRSTT in complex with its cognate tRNA shows that these twobases specifically interact with the enzyme, whereas base 34, which can beany base, is stacked under base 33 and makes no interactions with thesynthetase. This is in agreement with biochemical experiments whichidentify bases 35 and 36 as major tRNA identity elements. In contrast,class IIb synthetases (AspRS, AsnRS and LysRS) have a distinctanticodon-binding domain that specifically recognises all three anticodonbases. This again correlates with the requirements of the genetic code forcognate tRNA identification, as the class IIb amino acids occupy halfcodon groups.

• Brunel C et al.
• Translational regulation of the Escherichia coli threonyl-tRNA synthetasegene: structural and functional importance of the thrS operator domains.
• Biochimie. 1993; 75: 1167-79
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• Previous work showed that E coli threonyl-tRNA synthetase (ThrRS) binds tothe leader region of its own mRNA and represses its translation byblocking ribosome binding. The operator consists of four distinct domains,one of them (domain 2) sharing structural analogies with the anticodon armof the E coli tRNA(Thr). The regulation specificity can be switched byusing tRNA identity rules, suggesting that the operator could berecognized by ThrRS as a tRNA-like structure. In the present paper, weinvestigated the relative contribution of the four domains to theregulation process by using deletions and point mutations. This wasachieved by testing the effects of the mutations on RNA conformation (byprobing experiments), on ThrRS recognition (by footprinting experimentsand measure of the competition with tRNA(Thr) for aminoacylation), onribosome binding and ribosome/ThrRS competition (by toeprintingexperiments). It turns out that: i) the four domains are structurally andfunctionally independent; ii) domain 2 is essential for regulation andcontains the major structural determinants for ThrRS binding; iii) domain4 is involved in control and ThrRS recognition, but to a lesser degreethan domain 2. However, the previously described analogies with theacceptor-like stem are not functionally significant. How it is recognizedby ThrRS remains to be resolved; iv) domain 1, which contains the ribosomeloading site, is not involved in ThrRS recognition. The binding of ThrRSprobably masks the ribosome binding site by steric hindrance and not bydirect contacts. This is only achieved when ThrRS interacts with bothdomains 2 and 4; and v) the unpaired domain 3, which connects domains 2and 4, is not directly involved in ThrRS recognition. It should serve asan articulation to provide an appropriate spacing between domains 2 and 4.Furthermore, it is possibly involved in ribosome binding.

• Kim S, Landro JA, Gale AJ, Schimmel P
• C-terminal peptide appendix in a class I tRNA synthetase needed foracceptor-helix contacts and microhelix aminoacylation.
• Biochemistry. 1993; 32: 13026-31
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• The 10 class I tRNA synthetases have an N-terminal nucleotide-binding foldwhich contains the catalytic center. Insertions into thenucleotide-binding fold provide contacts for acceptor-helix interactions,which stabilize the amino acid acceptor end of the tRNA substrate in theactive site. A separate and largely nonconserved C-terminal domainprovides contacts with distal parts of the tRNA, including the anticodon.For Escherichia coli methionyl tRNA synthetase, whose structure is known,the C-terminal domain is predominantly alpha-helical and forms a loopwhich interacts with the anticodon trinucleotide located about 76 A fromthe amino acid attachment site. Fused to the end of this helical domain isa peptide which curls back into the N-terminal nucleotide-binding fold andregion of the active site. We show here that mutations in this peptideappendix disrupt aminoacylation and binding of a 7 base pair microhelixsubstrate based on the acceptor stem of tRNA(fMet), without affectinginteractions with ATP or methionine or with the tRNA(fMet) anticodon. Theimpairment of acceptor-helix interactions by mutation of the C-terminalpeptide can offset favorable anticodon interactions and severely reduceaminoacylation of tRNA(fMet). Thus, in addition to, or as an alternativeto, acceptor-helix-binding insertions into the N-terminalnucleotide-binding fold, C-terminal peptide epitopes in some class Ienzymes may provide a mechanism for facilitating RNA microhelixinteractions with the catalytic site.

• Dyson MR, Mandal N, RajBhandary UL
• Relationship between the structure and function of Escherichia coliinitiator tRNA.
• Biochimie. 1993; 75: 1051-60
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• Through functional studies of mutant tRNAs, we have identified sequenceand/or structural features important for specifying the many distinctiveproperties of E coli initiator tRNA. Many of the mutant tRNAs contain ananticodon sequence change from CAU-->CUA and are now substrates for E coliglutaminyl-tRNA synthetase (GlnRS). We describe here the effect of furthermutating the discriminator base 73 and nucleotide 72 at the end of theacceptor stem on: i) recognition of the mutant tRNAs by E coli GlnRS; ii)recognition by E coli methionyl-tRNA transformylase; and iii) activity ofthe mutant tRNAs in initiation in E coli. For GlnRS recognition, ourresults are, in general, consistent with interactions found in the crystalstructure of the E coli GlnRS-glutamine tRNA complex. The results alsosupport our previous conclusion that formylation of initiator tRNA isimportant for its function in initiation.

• Romby P et al.
• Molecular mimicry in translational control of E. coli threonyl-tRNAsynthetase gene. Competitive inhibition in tRNA aminoacylation andoperator-repressor recognition switch using tRNA identity rules.
• Nucleic Acids Res. 1992; 20: 5633-40
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• We previously showed that: (i) E.coli threonyl-tRNA synthetase (ThrRS)binds to the leader of its mRNA and represses translation by preventingribosome binding to its loading site; (ii) the translational operatorshares sequence and structure similarities with tRNA(Thr); (iii) it ispossible to switch the specificity of the translational control from ThrRSto methionyl-tRNA synthetase (MetRS) by changing the CGU anticodon-likesequence to CAU, the tRNA(Met) anticodon. Here, we show that the wild type(CGU) and the mutated (CAU) operators act as competitive inhibitors oftRNA(Thr) and tRNA(fMet) for aminoacylation catalyzed by E.coli ThrRS andMetRS, respectively. The apparent Kd of the MetRS/CAU operator complex isone order magnitude higher than that of the ThrRS/CGU operator complex.Although ThrRS and MetRS shield the anticodon- and acceptor-like domainsof their respective operators, the relative contribution of these twodomains differs significantly. As in the threonine system, the interactionof MetRS with the CAU operator occludes ribosome binding to its loadingsite. The present data demonstrate that the anticodon-like sequence is onemajor determinant for the identity of the operator and the regulationspecificity. It further shows that the tRNA-like operator obeys to tRNAidentity rules.

• Rould MA, Perona JJ, Soll D, Steitz TA
• Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln)and ATP at 2.8 A resolution.
• Science. 1989; 246: 1135-42
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• The crystal structure of Escherichia coli glutaminyl-tRNA synthetase(GlnRS) complexed with its cognate glutaminyl transfer RNA (tRNA(Gln] andadenosine triphosphate (ATP) has been derived from a 2.8 angstromresolution electron density map and the known protein and tRNA sequences.The 63.4-kilodalton monomeric enzyme consists of four domains arranged togive an elongated molecule with an axial ratio greater than 3 to 1. Itsinteractions with the tRNA extend from the anticodon to the acceptor stemalong the entire inside of the L of the tRNA. The complexed tRNA retainsthe overall conformation of the yeast phenylalanine tRNA (tRNA(Phe] withtwo major differences: the 3' acceptor strand of tRNA(Gln) makes a hairpinturn toward the inside of the L, with the disruption of the final basepair of the acceptor stem, and the anticodon loop adopts a conformationnot seen in any of the previously determined tRNA structures. Specificrecognition elements identified so far include (i) enzyme contacts withthe 2-amino groups of guanine via the tRNA minor groove in the acceptorstem at G2 and G3; (ii) interactions between the enzyme and the anticodonnucleotides; and (iii) the ability of the nucleotides G73 and U1.A72 ofthe cognate tRNA to assume a conformation stabilized by the protein at alower free energy cost than noncognate sequences. The central domain ofthis synthetase binds ATP, glutamine, and the acceptor end of the tRNA aswell as making specific interactions with the acceptor stem.2+t is