Zinc ion mediated amino acid recognition by threonyl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) ensure faithful translation of mRNA into protein by coupling an amino acid to a set of tRNAs with conserved anticodon sequences. Here, we show that in mitochondria of Saccharomyces cerevisiae, a single aaRS (MST1) recognizes and aminoacylates two natural tRNAs that contain anticodon loops of different size and sequence. Besides a regular tRNA Thr 2 with a threonine (Thr) anticodon, MST1 also recognizes an unusual tRNA Thr 1 , which contains an enlarged anticodon loop and an anticodon triplet that reassigns the CUN codons from leucine to threonine. Our data show that MST1 recognizes the anticodon loop in both tRNAs, but employs distinct recognition mechanisms. The size but not the sequence of the anticodon loop is critical for tRNA Thr 1 recognition, whereas the anticodon sequence is essential for aminoacylation of tRNA Thr 2 . The crystal structure of MST1 reveals that, while lacking the N-terminal editing domain, the enzyme closely resembles the bacterial threonyl-tRNA synthetase (ThrRS). A detailed structural comparison with Escherichia coli ThrRS, which is unable to aminoacylate tRNA Thr 1 , reveals differences in the anticodon-binding domain that probably allow recognition of the distinct anticodon loops. Finally, our mutational and modeling analyses identify the structural elements in MST1 (e.g., helix α11) that define tRNA selectivity. Thus, MTS1 exemplifies that a single aaRS can recognize completely divergent anticodon loops of natural isoacceptor tRNAs and that in doing so it facilitates the reassignment of the genetic code in yeast mitochondria.protein synthesis | anticodon recognition

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“…active-site groove as previously suggested (29). However, this mechanism does not explain the basis for the rejection of serine.…”

Section: Modeling Of the Mst1-trna Thr Complex And Mutational Study Smentioning

confidence: 62%

Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

Ling

Peterson

Simonović

et al. 2012

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

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“…In the presence of a wild-type chromosomal copy of thrS, the thrS-lacZ fusion synthesizes 1,339 Miller units of ␤-galactosidase when wild-type thrS expression is not induced from plasmid pTetthrS⌬H ( Table 2). This level dropped to 25 Miller units under induced conditions, giving a repression factor of 53. Because of the dominant lethal effects, steady-state exponential growth is not achievable with the pTetthrS⌬H plasmids carrying mutant versions of thrS under induced conditions.…”

Section: Resultsmentioning

confidence: 99%

“…For many aminoacyl-tRNA synthetases, the zinc atom has a structural role, shaping the enzyme in a three-dimensional structure compatible with its function (20). In ThrRS, the zinc atom is involved directly in threonine recognition by interacting with both its amino and hydroxyl groups (25,26). The direct involvement of the zinc atom in amino acid recognition was recently shown in another case, that of seryltRNA synthetase from a methanogenic archaea (1).…”

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confidence: 99%

Mutations in Residues Involved in Zinc Binding in the Catalytic Site of Escherichia coli Threonyl-tRNA Synthetase Confer a Dominant Lethal Phenotype

et al. 2007

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Escherichia coli threonyl-tRNA synthetase is a homodimeric protein that acts as both an enzyme and a regulator of gene expression: the protein aminoacylates tRNA Thr isoacceptors and binds to its own mRNA, inhibiting its translation. The enzyme contains a zinc atom in its active site, which is essential for the recognition of threonine. Mutations in any of the three amino acids forming the zinc-binding site inactivate the enzyme and have a dominant negative effect on growth if the corresponding genes are placed on a multicopy plasmid. We show here that this particular property is not due to the formation of inactive heterodimers, the titration of tRNA Thr by an inactive enzyme, or its misaminoacylation but is, rather, due to the regulatory function of threonyl-tRNA synthetase. Overproduction of the inactive enzyme represses the expression of the wild-type chromosomal copy of the gene to an extent incompatible with bacterial growth.

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“…These transcripts include one that skips exon 6, another that skips exons 6 and 7, and a third that terminates after exon 6. A fourth transcript encodes AlaXp (exons [9][10][11][12][13][14][15][16][17][18][19][20]. Finally, the remaining two transcripts are special fusions of coding sequences for p23 H with those for AlaXp.…”

mentioning

confidence: 99%

“…The Ser/Ala confusion is surprising, because serine is larger than alanine and, for other tRNA synthetases, the challenge is to eliminate similar or smaller, but not larger, amino acids from the active site. Among other examples (5-7) are misactivation of valine by IleRS (8)(9)(10), glycine by AlaRS (1), valine by LeuRS (11), and serine by ThrRS (12). The misactivated amino acids (in the form of the aminoacyl adenylate or as the mischarged tRNA, such as Val-tRNA Ile ) are removed by hydrolytic editing, using a second active site (13)(14)(15).…”

mentioning

confidence: 99%

p23 ^H implicated as cis/trans regulator of AlaXp-directed editing for mammalian cell homeostasis

Nawaz

Merriman

Yang

et al. 2011

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

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The toxicity of mistranslation of serine for alanine appears to be universal, and is prevented in part by the editing activities of alanyl-tRNA synthetases (AlaRSs), which remove serine from mischarged tRNA Ala . The problem of serine mistranslation is so acute that free-standing, genome-encoded fragments of the editing domain of AlaRSs are found throughout evolution. These AlaXps are thought to provide functional redundancy of editing. Indeed, archaeal versions rescue the conditional lethality of bacterial cells harboring an editing-inactive AlaRS. In mammals, AlaXps are encoded by a gene that fuses coding sequences of a homolog of the HSP90 cochaperone p23 (p23 H ) to those of AlaXp, to create p23 H AlaXp. Not known is whether this fusion protein, or various potential splice variants, are expressed as editing-proficient proteins in mammalian cells. Here we show that both p23 H AlaXp and AlaXp alternative splice variants can be detected as proteins in mammalian cells. The variant that ablated p23 H and encoded just AlaXp was active in vitro. In contrast, neither the p23 H AlaXp fusion protein, nor the mixture of free p23 H with AlaXp, was active. Further experiments in a mammalian cell-based system showed that RNAi-directed suppression of sequences encoding AlaXp led to a serine-sensitive increase in the accumulation of misfolded proteins. The results demonstrate the dependence of mammalian cell homeostasis on AlaXp, and implicate p23 H as a cis - and trans -acting regulator of its activity.

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Zinc ion mediated amino acid recognition by threonyl-tRNA synthetase

Abstract: ECM Nancy s93 conference abstracts s8a.m7.o3 Dioxygen reduction in strict anaerobes. Crystal structure of rubredoxin:oxygen oxidoreductase (ROO) from Desulfovibrio gigas.

Cited by 32 publications

References 2 publications

Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

Mutations in Residues Involved in Zinc Binding in the Catalytic Site of Escherichia coli Threonyl-tRNA Synthetase Confer a Dominant Lethal Phenotype

p23 ^H implicated as cis/trans regulator of AlaXp-directed editing for mammalian cell homeostasis

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Zinc ion mediated amino acid recognition by threonyl-tRNA synthetase

Abstract: ECM Nancy s93 conference abstracts s8a.m7.o3 Dioxygen reduction in strict anaerobes. Crystal structure of rubredoxin:oxygen oxidoreductase (ROO) from Desulfovibrio gigas.

Cited by 32 publications

References 2 publications

Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

Mutations in Residues Involved in Zinc Binding in the Catalytic Site of Escherichia coli Threonyl-tRNA Synthetase Confer a Dominant Lethal Phenotype

p23 H implicated as cis/trans regulator of AlaXp-directed editing for mammalian cell homeostasis

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p23 ^H implicated as cis/trans regulator of AlaXp-directed editing for mammalian cell homeostasis