Expression of Escherichia coli Methionyl-tRNA Formyltransferase in Saccharomyces cerevisiae Leads to Formylation of the Cytoplasmic Initiator tRNA and Possibly to Initiation of Protein Synthesis with Formylmethionine

Abstract

Protein synthesis in eukaryotic cytoplasm and in archaebacteria is initiated with methionine, whereas, that in eubacteria and in eukaryotic organelles, such as mitochondria and chloroplasts, is initiated with formylmethionine. In view of this clear distinction, we have investigated whether protein synthesis in the eukaryotic cytoplasm can be initiated with formylmethionine, and, if so, what the consequences are to the cell. For this purpose, we have expressed in an inducible manner the Escherichia coli methionyl-tRNA formyltransferase (MTF) in the cytoplasm of the yeast Saccharomyces cerevisiae. Expression of active MTF, but not of an inactive mutant, leads to formylation of methionine attached to the yeast cytoplasmic initiator tRNA to the extent of about 70%. As a consequence, the yeast strain grows slowly. Coexpression of the E. coli polypeptide deformylase (DEF), which removes the formyl group from the N-terminal formylmethionine in a polypeptide, rescues the slow-growth phenotype, whereas, coexpression of an inactive mutant of DEF does not. These results suggest that the cytoplasmic protein-synthesizing system of yeast, like that of eubacteria, can at least to some extent utilize formylated initiator Met-tRNA to initiate protein synthesis and that initiation of proteins with formylmethionine leads to the slow-growth phenotype. Removal of the formyl group in these proteins by DEF would explain the rescue of the slow-growth phenotype.Protein synthesis in eubacteria, mitochondria, and chloroplasts is initiated with formylmethionine (20,21,41). Of the two classes of methionine tRNAs present in eubacteria, mitochondria, and chloroplasts, tRNA Met and tRNA f Met , the initiator methionyl-tRNA (Met-tRNA f Met ) is specifically formylated by the enzyme methionyl-tRNA formyltransferase (MTF) to generate formylmethionyl-tRNA (fMet-tRNA f Met ). The formyl group acts as a positive determinant for selection of the fMet-tRNA f Met by the initiation factor IF2 (54) and as a second negative determinant for blocking the binding of the tRNA to the elongation factor. Formylation of the initiator Met-tRNA f Met is important for protein synthesis in Escherichia coli, mutant initiator tRNAs defective in formylation are extremely poor in initiation of protein synthesis, and a strain of E. coli carrying disruptions in the MTF gene has severe growth defects (12,33,59).In contrast to the situation with eubacteria, mitochondria, and chloroplasts presented above, in the cytoplasm of eukaryotes and in archaebacteria, there is no formylation of the initiator tRNA (tRNA i Met ), and the Met-tRNA i Met is utilized directly to initiate protein synthesis with methionine (42, 47). However, the cytoplasmic initiator Met-tRNA i Met of yeast contains some of the determinants in the tRNA (Fig. 1) important for recognition by E. coli MTF (12a, 23) and can therefore be formylated in vitro by E. coli MTF (43). The resulting fMet-tRNA i Met initiates protein synthesis in cell extracts of both E. coli and rabbit reticulocytes, suggesting that the eu...

Understanding the Genetic Code

“…Initiation codons, acting with an initiation tRNA, usually encode formyl methionine (fMet) in bacteria, chloroplasts, and mitochondria or methionine (Met) in archaea and the cytosol of eukaryotes (34)(35)(36). The codon wobble position is P1, where the order of usage for prokaryotes is usually AUG Ͼ GUG Ͼ UUG Ͼ CUG.…”

Protein N-terminal methionine excision

“…Eubacterial and organellar nascent proteins begin with an N-formylated Met. Peptide deformylase (PDF; EC 3.5.1.88) activity is required to unmask the first Met, facilitating the action of MAP [21,22]. NME is an irreversible cotranslational mechanism, completed before the nascent polypeptide chains are fully synthesized [23].…”

The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases

“…Ramesh et al [14] showed that expression of E. coli MTF in S. cerevisiae led to formylation of the cytoplasmic initiator (up to 70%) and possibly to initiation of protein synthesis with formylmethionine. The slow growth phenotype of yeast caused by expression of E. coli MTF could be rescued by co-expressing E. coli polypeptide deformylase (DEF), an enzyme that removes formyl groups from the N-terminus of proteins.…”

“…various Halobacterial species [11]; Methanocaldococcus jannaschii; Methanococcus maripaludis [12]), yeast (Saccharomyces cerevisiae [13,14]), and mammalian cells (e.g. COS1, HEK293, HeLa cells [15][16][17][18]).…”