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Cytoplasmic methionyl-tRNA synthetase from Bakers' yeast. A monomer with a post-translationally modified N terminus
Abstract:
Methionyl-tRNA synthetase has been purified from a yeast strain carrying the MES1 structural gene on a high copy number plasmid (pFL1). The purified enzyme is a monomer of Mr = 85,000 in contrast to its counterpart from Escherichia coli which is a dimer made up of identical subunits (Mr = 76,000; Dardel, F., Fayat, G., and Blanquet, S. (1984) J. Bacteriol. 160, 1115-1122). The yeast enzyme was not amenable to Edman's degradation indicating a blocked NH2 terminus. Its primary structure as derived from the DNA sequence (Walter, P., Gangloff, J., Bonnet, J., Boulanger, Y., Ebel, J.P., and Fasiolo, F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2437-2441) has been confirmed using the fast atom bombardment-mass spectrometric method. This method was applied to tryptic digests of the carboxymethylated enzyme and the corresponding data provided extensive coverage of the translated DNA sequence, thus confirming its correctness. The ambiguity concerning which of the three NH2-terminally located methionine codons is the initiation codon was easily resolved from peptides identified in this region. It was possible to show that the first methionine had been removed and that the new NH2 terminus, serine, had been acetylated. A comparison between the yeast and E. coli sequences shows that the former has an N-terminal extension of about 200 residues as compared to the latter. It also lacks the C-terminal domain which is responsible for the dimerization of the E. coli methionyl-tRNA synthetase.
Insights
This study purified yeast methionyl-tRNA synthetase, revealing a monomeric structure distinct from its bacterial counterpart. Mass spectrometry confirmed the primary structure and identified an acetylated N-terminal serine.
Area of Science:
- Molecular Biology
- Biochemistry
- Enzymology
Background:
- Methionyl-tRNA synthetase is crucial for protein synthesis initiation.
- The enzyme's structure and function vary across species, notably between yeast and Escherichia coli.
- Previous studies established the DNA sequence of the yeast MES1 gene.
Purpose of the Study:
- To purify and characterize yeast methionyl-tRNA synthetase.
- To confirm the primary structure derived from the MES1 gene sequence.
- To compare the structural features of yeast methionyl-tRNA synthetase with its E. coli counterpart.
Main Methods:
- Purification of methionyl-tRNA synthetase from a yeast strain overexpressing the MES1 gene.
- Analysis of the enzyme's molecular weight and subunit composition (monomer vs. dimer).
- Fast atom bombardment-mass spectrometry (FAB-MS) of tryptic digests of the carboxymethylated enzyme to confirm primary structure and N-terminal modifications.
Main Results:
- Purified yeast methionyl-tRNA synthetase is a monomer (Mr = 85,000), unlike the dimeric E. coli enzyme.
- FAB-MS confirmed the DNA-derived primary structure and identified an acetylated N-terminal serine, indicating removal of the initiating methionine.
- Yeast enzyme possesses an N-terminal extension and lacks the C-terminal dimerization domain found in E. coli methionyl-tRNA synthetase.
Conclusions:
- The primary structure of yeast methionyl-tRNA synthetase, including N-terminal processing, is confirmed.
- Significant structural differences exist between yeast and E. coli methionyl-tRNA synthetases, particularly in subunit association and terminal domains.
- These structural variations likely contribute to functional differences in protein synthesis initiation between yeast and E. coli.