Distinct nuclear genes for yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases

Schneller, Jean-Marie; Schneider, Claude; Stahl, AndréJ.C.

doi:10.1016/0006-291x(78)91158-0

Cited by 18 publications

(8 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The present studies confirm earlier biochemical evidence that the mitochondrial and Cytoplasmic methionyl-tRNA synthetases of yeast are products of two separate nuclear genes [31]. The inability of mitochondrial extracts obtained from two different msml mutants to catalyze the acylation of both the elongator and initiator methionyl-tRNAs indicates that as in prokaryotic systems [32], both isoacceptors are charged by the same enzyme.…”

Section: Sequence Of the Msml Genesupporting

confidence: 89%

Characterization of MSM1, the structural gene for yeast mitochondrial methionyl‐tRNA synthetase

Tzagoloff

Vambutas

Akai

1989

European Journal of Biochemistry

View full text Add to dashboard Cite

Respiratory-deficient mutants of Saccharomyces cerevisiae assigned to p e t complementation group G72 are impaired in mitochondrial protein synthesis. The loss of this activity has been correlated with the inability of the mutants to acylate the two methionyl-tRNAs of yeast mitochondria. A nuclear gene (MSM1) capable of complementing the respiratory deficiency has been cloned by transformation of the G72 mutant C122/U3 with a yeast genomic library. In situ disruption of the M S M l gene in a wild-type haploid strain of yeast induces a respiratory-deficient phenotype but does not affect the ability of the mutant to grow on fermentable substrates indicating that the product of M S M l functions only in mitochondrial protein synthesis. Mitochondria1 extracts prepared from the mutant with the disrupted copy of MSMl were found to be defective in acylation of the two mitochondrial methionyl-tRNAs thereby confirming the identity of M S M l as the structural gene for the mitochondrial methionyl-tRNA synthetase. The sequence of the protein encoded by MSMI is similar to the Escherichia coli and yeast cytoplasmic methionyl-tRNA synthetases. Based on the primary-sequence similarities of the three proteins, the mitochondrial enzyme appears to be more related to the bacterial than to the yeast cytoplasmic methionyl-tRNA synthetase.Yeast mitochondria contain a complete set of aminoacyltRNA synthetases that catalyze the acylation of the 23 endogenous cognate tRNAs [l]. In contrast to the tRNA substrates whose genes are located in mitochondrial DNA [l, 21, the synthetases are all encoded by nuclear genes. Only the mitochondrial and cytoplasmic histidyl-and valyl-tRNA synthetases of yeast are presently known to be products of common genes [3,4]. All the other mitochondrial synthetases studied to date have been found to be encoded by genes distinct from those coding for the similar cytoplasmic enzymes As part of a study aimed at characterizing the aminoacyltRNA synthetases of mitochondria, we have screened a collection of nuclear respiratory-deficient mutants of yeast for strains defective in acylation of mitochondrial tRNAs [S]. In the present communication, we report the properties of a pet complementation group whose members are impaired in the acylation of the two mitochondrial methionyl-tRNAs. A gene capable of restoring the respiratory deficiency of a representative mutant from this complementation group has been cloned by transformation with a yeast genomic library. The cloned gene designated as M S M l codes for a protein with M , 66 558. The amino acid sequence of the MSMI product derived from the gene sequence is similar to the Escherichia coli [9] and yeast methionyl-tRNA synthetases [lo]. These results indicate that both mitochondrial methionyl-tRNA isoacceptors are acylated by a single enzyme whose gene is distinct from the gene coding for the cytoplasmic methionyl-tRNA synthetase.[S -81.Correspondence fo A. Tzagoloff, Department of Biological Sciences. Columbia University, New York, NY 10027, USA Ahhrevintions. pet, ...

show abstract

Section: Sequence Of the Msml Genesupporting

confidence: 89%

Characterization of MSM1, the structural gene for yeast mitochondrial methionyl‐tRNA synthetase

Tzagoloff

Vambutas

Akai

1989

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…To conclude, we emphasize that transformation to prototrophy of our yeast Km mutant could be due to the cloning of either cytoplasmic or mitochondrial methionyl-tRNA synthetase genes because both belong to the nuclear genome and both enzymes aminoacylate cytoplasmic tRNA (18). However, the two enzymes can be distinguished on the basis of two criteria (18): (i) their chromatographic behavior on hydroxyapatite columns, the mitochondrial enzyme being eluted at 0.1 M phosphate and the cytoplasmic one at 0.2 M; and (ii) the specificity of aminoacylation.…”

Section: Resultsmentioning

confidence: 99%

“…However, the two enzymes can be distinguished on the basis of two criteria (18): (i) their chromatographic behavior on hydroxyapatite columns, the mitochondrial enzyme being eluted at 0.1 M phosphate and the cytoplasmic one at 0.2 M; and (ii) the specificity of aminoacylation. Indeed, mitochondrial methionyl-tRNA synthetase aminoacylates both yeast cytoplasmic and E. coli tRNA at similar rates whereas its cytoplasmic counterpart aminoacylates E. coli tRNA at a rate 1/7th that of yeast cytoplasmic tRNA (18). We recently have found that antibodies raised against the purified gene product do not inhibit the mitochondrial methionyl-tRNA synthetase.…”

Section: Resultsmentioning

confidence: 99%

Primary structure of the Saccharomyces cerevisiae gene for methionyl-tRNA synthetase.

Walter¹,

Gangloff²,

Bonnet³

et al. 1983

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

View full text Add to dashboard Cite

The sequence of a 5-kilobase DNA insert containing the structural gene for yeast cytoplasmic methionyl-tRNA synthetase has been determined and a unique open reading frame of 2,253 nucleotides encoding a polypeptide chain of 751 amino acids (Mr, 85,5W) has been characterized. The data obtained on the purified enzyme (subunit size, amino acid composition, and COOH-terminal sequence) are consistent with the gene structure. The protein sequence deduced from the nucleotide sequence reveals no obvious internal repeats. This protein sequence shows a high degree of homology with that of Eacherichia coli methionyltRNA synthetase within a region that forms the putative methionyl adenylate binding site. This strongly suggests that both proteins derive from a common ancestor.Aminoacyl-tRNA synthetases play a crucial role in protein synthesis because they catalyze the specific attachment of amino acids to their cognate tRNAs. Knowledge of their primary structure is one of the prerequisites for the complete understanding of the structure-function relationship. So far, the sequence of only one aminoacyl-tRNA synthetase has been completely determined by using classical protein sequence analysis techniques (1). However, the cloning of a number of aminoacyltRNA synthetases genes has allowed derivation of the primary structure of the corresponding enzyme from the DNA sequence-i.e., the alanyl-(2), tryptophanyl-(3), glutaminyl-(4), and methionyl-(5) tRNA synthetases from Escherichia coli.In our laboratory, a yeast mutant strain lacking functional cytoplasmic methionyl-tRNA synthetase was available. It was complemented with a plasmid pool containing random fragments of wild-type yeast genomic DNA obtained by a partial Sau3A digestion (6). It was thus possible to isolate a 5. 1-kilobase piece of DNA containing the methionyl-tRNA synthetase gene (MES1). The isolated gene product is a monomer (Mr, 80,000). However, in crude extracts from a wild-type strain the enzyme behaves as a dimer (Mr, 2 x 80,000). Because enzyme purification always led to a monomeric species with no detectable variation of Mr, the existence of a dimeric structure for native methionyl-tRNA synthetase is by no means proven (unpublished data).In this paper we report the complete nucleotide sequence of the DNA insert containing the structural gene. Indeed, a unique open reading frame whose length corresponds exactly to that expected for the above protein size could be characterized.MATERIALS AND METHODS DNA Sequence Analysis. DNA sequence analysis was carried out by the chain termination method of Sanger et al. (7).Fragments of the cloned DNA were subeloned into the bacteriophage M13mp7 (8) and analyzed by using the primer synthesized by R. Crea and purchased from P-L Biochemicals.Enzymes and Chemicals. Most restriction enzymes and T4 DNA ligase were obtained from New England BioLabs. The Klenow E. coli polymerase and nuclease SI were from Boehringer Mannheim.All chemicals were from Merck (Darmstadt, Federal Republic of Germany) and were analytical grade. ...

show abstract

“…In particular, elongator tRNAMet from rabbit liver has been reported not to bind MTSEC better than a non-Met tRNA (17). In addition, yeast mitochondrial and wheat germ chloroplastic MTS aminoacylate as efficiently E. coli tRNAsMet as their cognate tRNAs (20)(21)(22). Finally, eukaryotic cytoplasmic MTS aminoacylate the mitochondrial, choroplastic and E.coli tRNAsMet with a low efficiency (20-22) whereas mammalian cytoplasmic MTS efficiently aminoacylates cytoplasmic tRNAMet from yeast (23).…”

Section: Introductionmentioning

confidence: 99%

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian andE.colimethionyl-tRNA synthetases

et al. 1992

View full text Add to dashboard Cite

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).

show abstract

Distinct nuclear genes for yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases

Cited by 18 publications

References 7 publications

Characterization of MSM1, the structural gene for yeast mitochondrial methionyl‐tRNA synthetase

Characterization of MSM1, the structural gene for yeast mitochondrial methionyl‐tRNA synthetase

Primary structure of the Saccharomyces cerevisiae gene for methionyl-tRNA synthetase.

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian andE.colimethionyl-tRNA synthetases

Contact Info

Product

Resources

About