Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L-Threonine by the Complex of tRNAVal and Valyl-tRNA Synthetase

Fukai, Shuya; Nureki, Osamu; Sekine, S.; Shimada, Akira; Tao, Jianshi; Vassylyev, Dmitry G.; Yokoyama, Shigeyuki

doi:10.1016/s0092-8674(00)00182-3

Cited by 263 publications

(322 citation statements)

References 42 publications

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“…In this situation, glycine could also be accommodated in the editing site, because it has no side chain that conflicts with Gln-584, although glycine should be tightly held by interactions of main-chain moiety, which are lacking in our AlaX structure. Similar inclusion of smaller amino acids has been shown in a class I valyl-tRNA synthetase editing site (20), showing that smaller noncognate ␣-aminobutyrate or cysteine could sterically be accommodated within the editing site, whereas a larger cognate, valine, is chemically excluded. Furthermore, we observed that the Q584M mutation slightly impairs the deacylation of Gly-tRNA Ala (data not shown), suggesting that the Gln-584 not only prevents the entry of an alanine side chain but also could interact with an ␣-hydrogen of glycine by so-called weak hydrogen bonding.…”

Section: Discussionsupporting

confidence: 59%

Molecular basis of alanine discrimination in editing site

Sokabe

Okada

Yao

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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AlaX is the homologue of the class II alanyl-tRNA synthetase editing domain and has been shown to exhibit autonomous editing activity against mischarged tRNA Ala . Here, we present the structures of AlaX from the archaeon Pyrococcus horikoshii in apo form, complexed with zinc, and with noncognate amino acid L-serine and zinc. Together with mutational analysis, we demonstrated that the conserved Thr-30 hydroxyl group located near the ␤-methylene of the bound serine is responsible for the discrimination of noncognate serine from cognate alanine, based on their chemical natures. Furthermore, we confirmed that the conserved Gln-584 in alanyl-tRNA synthetase, which corresponds to Thr-30 of AlaX, is also critical for discrimination. These observations strongly suggested conservation of the chemical discrimination among trans-and cis-editing of tRNA Ala .alanyl-tRNA synthetase ͉ class II tRNA synthetase ͉ crystal structure ͉ trans-editing A minoacyl-tRNA synthetases (aaRSs) establish the genetic code through aminoacylation of cognate tRNA (1). However, in some aaRSs, the affinity difference of the active site is not large enough to distinguish among similar amino acids with sufficient accuracy. Therefore, during evolution, an additional editing domain that specifically hydrolyzes mischarged tRNAs has assembled with the catalytic domain to comprise contemporary aaRSs (2). In accordance with this model, the genes that autonomously encode an editing domain were distributed in many organisms (3-5), and, indeed, some of them are shown to be responsible for the transediting activity of mischarged tRNAs (4, 5). AlaX is the one such protein that shows homology to the class II alanyl-tRNA synthetase (AlaRS) editing domain ( Fig. 1) and is widely scattered among all three kingdoms of life (3). The specific activities of the archaeal AlaXs from Methanosarcina barkeri and Sulfolobus solfataricus have recently been shown to specifically hydrolyze mischarged Ser-and Gly-tRNA Ala in vitro (4).In contrast to the well established class I aaRSs, information regarding editing of the evolutionarily distinct class II aaRSs (to which AlaRS belongs) has only recently begun to emerge. The editing mechanism of the threonyl system has been investigated by structural analyses of the bacterial threonyl-tRNA synthetase (ThrRS) editing domain (hereafter called ThrRS-N2) complexed with the serine product or with the substrate analogue seryl-3Ј-aminoadenosine (SerA76) (6). These analyses showed that (i) cognate threonine and noncognate serine are discriminated by steric exclusion of the additional ␥-methyl group of threonine by the conserved His-77, Tyr-104, and Asp-180, and (ii) the HXXXH and CXXXH motifs characteristic of ThrRS-N2 should not bind a zinc ion for catalysis, despite the capability to bind zinc (7). The AlaX͞AlaRS editing domains are evolutionarily related to ThrRS-N2 and share the characteristic HXXXH and CXXXH motifs (Fig. 1) (4, 8), which were also shown to be important for the deacylation activity of AlaRS (9). The inclusion of non...

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Section: Discussionsupporting

confidence: 59%

Molecular basis of alanine discrimination in editing site

Sokabe

Okada

Yao

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…Although human GlyRS-IIA lacks cysteine ligands for Zn binding, the fold of the homologous region of the protein is maintained, so a model of the conserved Pfu GlyRS-IIA Zn-binding domain was obtained. Thermus thermophilus ( Tth ) ValRS-IA (PDB 1GAX) includes a shortened version of the shared Zn-binding domain [29]. In Figure 4, the shared Zn-binding regions are compared.…”

Section: Resultsmentioning

confidence: 99%

Aminoacyl-tRNA synthetase evolution and sectoring of the genetic code

2018

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The genetic code sectored via tRNA charging errors, and the code progressed toward closure and universality because of evolution of aminoacyl-tRNA synthetase (aaRS) fidelity and translational fidelity mechanisms. Class I and class II aaRS folds are identified as homologs. From sequence alignments, a structurally conserved Zn-binding domain common to class I and class II aaRS was identified. A model for the class I and class II aaRS alternate folding pathways is posited. Five mechanisms toward code closure are highlighted: 1) aaRS proofreading to remove mischarged amino acids from tRNA; 2) accurate aaRS active site specification of amino acid substrates; 3) aaRS-tRNA anticodon recognition; 4) conformational coupling proofreading of the anticodon-codon interaction; and 5) deamination of tRNA wobble adenine to inosine. In tRNA anticodons there is strong wobble sequence preference that results in a broader spectrum of contacts to synonymous mRNA codon wobble bases. Adenine is excluded from the anticodon wobble position of tRNA unless it is modified to inosine. Uracil is generally preferred to cytosine in the tRNA anticodon wobble position. Because of wobble ambiguity when tRNA reads mRNA, the maximal coding capacity of the three nucleotide code read by tRNA is 31 amino acids + stops.

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“…However, minihelices Leu cannot be mischarged, which may be explained by tRNA-independent pretransfer editing of ␣␤-LeuRS. For most known aaRS editing systems, both pretransfer and posttransfer editing are induced by the binding of tRNA to facilitate an editing complex conformation for aaRS even for pretransfer editing, which has no covalent linkage with tRNA (8,9,(47)(48)(49). However, some reports show that E. coli ProRS displays tRNA-independent pretransfer editing and yeast cytoplasmic LeuRS exhibits only pretransfer editing (36,50).…”

Section: Discussionmentioning

confidence: 99%

“…two active sites. Therefore, misactivated amino acids (pretransfer editing substrates) are translocated (8,9,51,52). However, the Thermus thermophilius LeuRS structure reveals a different scene, where the editing pocket has faced toward the aminoacylation pocket even in the absence of tRNA (10,53).…”

Section: Discussionmentioning

confidence: 99%

Leucyl-tRNA Synthetase from the Hyperthermophilic Bacterium Aquifex aeolicus Recognizes Minihelices

Zhao

Wang

2004

Journal of Biological Chemistry

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Aminoacylation of the minihelix mimicking the amino acid acceptor arm of tRNA has been demonstrated in more than 10 aminoacyl-tRNA synthetase systems. Although Escherichia coli or Homo sapiens cytoplasmic leucyl-tRNA synthetase (LeuRS) is unable to charge the cognate minihelix or microhelix, we show here that minihelix Leu is efficiently charged by Aquifex aeolicus synthetase, the only known heterodimeric LeuRS (␣␤-LeuRS). Aminoacylation of minihelices is strongly dependent on the presence of the A73 identity nucleotide and greatly stimulated by destabilization of the first base pair as reported for the E. coli isoleucyl-tRNA synthetase and methionyl-tRNA synthetase systems. In the E. coli LeuRS system, the anticodon of tRNA Leu is not important for recognition by the synthetase. However, the addition of RNA helices that mimic the anticodon domain stimulates minihelix Leu charging by ␣␤-LeuRS, indicating possible domain-domain communication within ␣␤-LeuRS. The leucine-specific domain of ␣␤-LeuRS is responsible for minihelix recognition. To ensure accurate translation of the genetic code, LeuRS functions to hydrolyze misactivated amino acids (pretransfer editing) and misaminoacylated tRNA (posttransfer editing). In contrast to tRNA Leu , minihelix Leu is unable to induce posttransfer editing even upon the addition of the anticodon domain of tRNA. Therefore, the context of tRNA is crucial for the editing of mischarged products. However, the minihelix Leu cannot be misaminoacylated, perhaps because of the tRNA-independent pretransfer editing activity of ␣␤-LeuRS.Aminoacyl-tRNA synthetases (aaRSs) 1 establish the genetic code by catalyzing the esterification of cognate amino acids to their specific transfer RNAs (tRNA) that bear the corresponding anticodons, which are defined by the genetic code (1). Aminoacylation of tRNA catalyzed by aaRSs is a two-step reaction: (a) activation of amino acids with ATP by formation of aminoacyl adenylate and (b) transfer of the aminoacyl moiety from the aminoacyl adenylate to the cognate tRNA substrate. On the basis of the architecture of the conserved active site domain, aaRSs are divided into two groups (2). Class I enzymes have an active site based on the Rossmann fold, whereas the conserved active core of class II enzymes contains an antiparallel ␤-sheet flanked by ␣-helices.One hypothesis about the assembly of aminoacyl-tRNA synthetases is that the enzymes are assembled in a modular fashion, incorporating new domains around the conserved active site representing ancestral aaRS such as the tRNA-binding domain and editing domain (3). Interestingly, two domains of tRNA (specifically the amino acid-accepting stem (minihelix) and anticodon stem biloop (SBL)), which interact with the aminoacylation active site and tRNA-binding domain of aaRS, respectively, may have arisen independently (4). Chargeable minihelices and smaller helices (e.g. microhelices of 7 bp) mimicking the acceptor stem have been identified in more than 10 aaRS systems (5). Thus, whereas it is recognized that aaRS...

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Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L-Threonine by the Complex of tRNAVal and Valyl-tRNA Synthetase

Cited by 263 publications

References 42 publications

Molecular basis of alanine discrimination in editing site

Molecular basis of alanine discrimination in editing site

Aminoacyl-tRNA synthetase evolution and sectoring of the genetic code

Leucyl-tRNA Synthetase from the Hyperthermophilic Bacterium Aquifex aeolicus Recognizes Minihelices

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