Yeast aspartyl-tRNA synthetase residues interacting with tRNAAsp identity bases connectively contribute to tRNAAsp binding in the ground and transition-state complex and discriminate against non-cognate tRNAs

International audienceCrystallographic studies of the aspartyl-tRNA synthetase-tRNAAsp complex from yeast identified on the enzyme a number of residues potentially able to interact with tRNAAsp. Alanine replacement of these residues (thought to disrupt the interactions) was used in the present study to evaluate their importance in tRNAAsp recognition and acylation. The results showed that contacts with the acceptor A of tRNAAsp by amino acid residues interacting through their side-chain occur only in the acylation transition state, whereas those located near the G73 discriminator base occur also during initial binding of tRNAAsp. Interactions with the anticodon bases provide the largest free energy contribution to stability of the enzyme-tRNA complex in its ground state. These contacts also favour catalysis, by acting connectively with each other and with those of G73, as shown by multiple mutant analysis. This implies structural communication transmitting the anticodon recognition signal to the distally located acylation site. This signal might be conveyed via tRNAAspas suggested by the observed conformational change of this molecule upon interaction with AspRS. From binding free energy values corresponding to the different AspRS-tRNAAsp interaction domains, it might be concluded that upon complex formation, the anticodon interacts first. Finally, acylation efficiencies of AspRS mutants in the presence of pure tRNAAsp and non-fractionated tRNAs indicate that residues involved in the binding of identity bases also discriminate against non-cognate tRNAs

Genetic selection for active E.coIi amber tRNAASn exclusively led to glutamine inserting suppressors

International audienceSuppressor tRNAs are useful tools for determining identity elements which define recognition of tRNAs in vivo by their cognate aminoacyl-tRNA synthetases. This study was aimed at the isolation of active amber tRNAASn. Nineteen mutated tRNAASnfcuA having amber suppressor activity were selected by an in vivo genetic screen, and all exclusively inserted glutamine. From analysis of the different mutations it is concluded that glutamine accepting activity was obtained upon reducing the interaction strength between the first base pair of the tRNAASnfCuA by direct or indirect effects. Failure to isolate tRNAASnfcuA suppressors charged with asparagine as well as other evolutionary related amino acids is discussed

A Gene Encoding Arginyl-tRNA Synthetase Is Located in the Upstream Region of the lysA Gene in Brevibacterium lactofermentum: Regulation of argS-lysA Cluster Expression by Arginine

International audienceThe Brevibacterium lactofermentum argS gene, which encodes an arginyl-tRNA synthetase, was identified in the upstream region of the lysA gene. The cloned gene was sequenced; it encodes a 550-amino-acid protein with an Mr of 59,797. The deduced amino acid sequence showed 28% identical and 49% similar residues when compared with the sequence of the Escherichia coli arginyl-tRNA synthetase. The B. lactofermentum enzyme showed the highly conserved motifs of class I aminoacyl-tRNA synthetases. Expression of the argS gene in B. lactofermentum and E. coli resulted in an increase in aminoacyl-tRNA synthetase activity, correlated with the presence in sodium dodecyl sulfate-polyacrylamide gels of a clear protein band that corresponds to this enzyme. One single transcript of about 3,000 nucleotides and corresponding to the B. lactofermentum argS-lysA operon was identified. The transcription of these genes is repressed by lysine and induced by arginine, showing an interesting pattern of biosynthetic interlock between the pathways of both amino acids in corynebacteria

Nucleic Acids Res

The leucine-specific domain (LSD) is a compact well-ordered module that participates in positioning of the conserved KMSKS catalytic loop in most leucyl-tRNA synthetases (LeuRSs). However, the LeuRS from Mycoplasma mobile (MmLeuRS) has a tetrapeptide GKDG instead of the LSD. Here, we show that the tetrapeptide GKDG can confer tRNA charging and post-transfer editing activity when transplanted into an inactive Escherichia coli LeuRS (EcLeuRS) that has had its LSD deleted. Reciprocally, the LSD, together with the CP1-editing domain of EcLeuRS, can cooperate when inserted into the scaffold of the minimal MmLeuRS, and this generates an enzyme nearly as active as EcLeuRS. Further, we show that LSD participates in tRNALeu recognition and favours the binding of tRNAs harbouring a large loop in the variable arm. Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding. Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD. The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD

Nucleic Acids Res

The fidelity of protein biosynthesis requires the aminoacylation of tRNA with its cognate amino acid catalyzed by aminoacyl-tRNA synthetase with high levels of accuracy and efficiency. Crucial bases in tRNA(Leu) to aminoacylation or editing functions of leucyl-tRNA synthetase have been extensively studied mainly by in vitro methods. In the present study, we constructed two Saccharomyces cerevisiae tRNA(Leu) knockout strains carrying deletions of the genes for tRNA(Leu)(GAG) and tRNA(Leu)(UAG). Disrupting the single gene encoding tRNA(Leu)(GAG) had no phenotypic consequence when compared to the wild-type strain. While disrupting the three genes for tRNA(Leu)(UAG) had a lethal effect on the yeast strain, indicating that tRNA(Leu)(UAG) decoding capacity could not be compensated by another tRNA(Leu) isoacceptor. Using the triple tRNA knockout strain and a randomly mutated library of tRNA(Leu)(UAG), a selection to identify critical tRNA(Leu) elements was performed. In this way, mutations inducing in vivo decreases of tRNA levels or aminoacylation or editing ability by leucyl-tRNA synthetase were identified. Overall, the data showed that the triple tRNA knockout strain is a suitable tool for in vivo studies and identification of essential nucleotides of the tRNA

Binding of human SLBP on the 3′-UTR of histone precursor H4-12 mRNA induces structural rearrangements that enable U7 snRNA anchoring

In metazoans, cell-cycle-dependent histones are produced from poly(A)-lacking mRNAs. The 3′ end of histone mRNAs is formed by an endonucleolytic cleavage of longer precursors between a conserved stem–loop structure and a purine-rich histone downstream element (HDE). The cleavage requires at least two trans-acting factors: the stem–loop binding protein (SLBP), which binds to the stem–loop and the U7 snRNP, which anchors to histone pre-mRNAs by annealing to the HDE. Using RNA structure-probing techniques, we determined the secondary structure of the 3′-untranslated region (3′-UTR) of mouse histone pre-mRNAs H4–12, H1t and H2a–614. Surprisingly, the HDE is embedded in hairpin structures and is therefore not easily accessible for U7 snRNP anchoring. Probing of the 3′-UTR in complex with SLBP revealed structural rearrangements leading to an overall opening of the structure especially at the level of the HDE. Electrophoretic mobility shift assays demonstrated that the SLBP-induced opening of HDE actually facilitates U7 snRNA anchoring on the histone H4–12 pre-mRNAs 3′ end. These results suggest that initial binding of the SLBP functions in making the HDE more accessible for U7 snRNA anchoring

Recognition of tRNALeu by Aquifex aeolicus leucyl-tRNA synthetase during the aminoacylation and editing steps

Recognition of tRNA by the cognate aminoacyl-tRNA synthetase during translation is crucial to ensure the correct expression of the genetic code. To understand tRNALeu recognition sets and their evolution, the recognition of tRNALeu by the leucyl-tRNA synthetase (LeuRS) from the primitive hyperthermophilic bacterium Aquifex aeolicus was studied by RNA probing and mutagenesis. The results show that the base A73; the core structure of tRNA formed by the tertiary interactions U8–A14, G18–U55 and G19–C56; and the orientation of the variable arm are critical elements for tRNALeu aminoacylation. Although dispensable for aminoacylation, the anticodon arm carries discrete editing determinants that are required for stabilizing the conformation of the post-transfer editing state and for promoting translocation of the tRNA acceptor arm from the synthetic to the editing site

Nucleic Acids Res

Aminoacyl-tRNA synthetases (aaRSs) are remarkable enzymes that are in charge of the accurate recognition and ligation of amino acids and tRNA molecules. The greatest difficulty in accurate aminoacylation appears to be in discriminating between highly similar amino acids. To reduce mischarging of tRNAs by non-cognate amino acids, aaRSs have evolved an editing activity in a second active site to cleave the incorrect aminoacyl-tRNAs. Editing occurs after translocation of the aminoacyl-CCA(76) end to the editing site, switching between a hairpin and a helical conformation for aminoacylation and editing. Here, we studied the consequence of nucleotide changes in the CCA(76) accepting end of tRNA(Leu) during the aminoacylation and editing reactions. The analysis showed that the terminal A(76) is essential for both reactions, suggesting that critical interactions occur in the two catalytic sites. Substitutions of C(74) and C(75) selectively decreased aminoacylation keeping nearly unaffected editing. These mutations might favor the regular helical conformation required to reach the editing site. Mutating the editing domain residues that contribute to CCA(76) binding reduced the aminoacylation fidelity leading to cell-toxicity in the presence of non-cognate amino acids. Collectively, the data show how protein synthesis quality is controlled by the CCA(76) homogeneity of tRNAs

Modular pathways for editing non-cognate amino acids by human cytoplasmic leucyl-tRNA synthetase

To prevent potential errors in protein synthesis, some aminoacyl-transfer RNA (tRNA) synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Class Ia leucyl-tRNA synthetase (LeuRS) may misactivate various natural and non-protein amino acids and then mischarge tRNALeu. It is known that the fidelity of prokaryotic LeuRS depends on multiple editing pathways to clear the incorrect intermediates and products in the every step of aminoacylation reaction. Here, we obtained human cytoplasmic LeuRS (hcLeuRS) and tRNALeu (hctRNALeu) with high activity from Escherichia coli overproducing strains to study the synthetic and editing properties of the enzyme. We revealed that hcLeuRS could adjust its editing strategy against different non-cognate amino acids. HcLeuRS edits norvaline predominantly by post-transfer editing; however, it uses mainly pre-transfer editing to edit α-amino butyrate, although both amino acids can be charged to tRNALeu. Post-transfer editing as a final checkpoint of the reaction was very important to prevent mis-incorporation in vitro. These results provide insight into the modular editing pathways created to prevent genetic code ambiguity by evolution