Formylation Is Not Essential for Initiation of Protein Synthesis in All Eubacteria

Abstract

Formylation of the initiator methionyl-tRNA, catalyzed by methionyl-tRNA formyltransferase, has long been regarded as essential for initiation of protein synthesis in eubacteria. Here, we show that this process is, in fact, dispensable in Pseudomonas aeruginosa. Disruption of the chromosomal methionyl-tRNA formyltransferase gene in P. aeruginosa resulted only in a moderate decrease in the rate of cell growth, whereas in Escherichia coli cell growth was severely impaired. The ability of the P. aeruginosa mutant strain to grow was not due to an additional copy of the methionyl-tRNA formyltransferase gene or to N-acylation of the methionyl moiety by a group other than formyl. These results indicate that P. aeruginosa can carry out formylation-independent initiation of protein synthesis, using the nonformylated methionyl-tRNA. Therefore, the dogma that eubacteria require formylation of the initiator methionyltRNA for initiation of protein synthesis may have been an invalid generalization of results obtained with E. coli.Protein synthesis in eubacteria, and in the chloroplasts and mitochondria of eukaryotes, can be initiated using the initiator formyl-methionyl-tRNA (fMet-tRNA fMet ) 1 (1). Formylation is specific for the initiator methionyl-tRNA (Met-tRNA fMet ) and is catalyzed by methionyl-tRNA formyltransferase (MTF), which is encoded by the fmt gene (2-5). The features in the Escherichia coli tRNA fMet required for formylation are the base-base mismatch between nucleotides 1 and 72 and the second and third base pairs of the acceptor stem (4 -6). The fMet moiety allows initiation factor IF-2 to recognize the initiator tRNA and reject other tRNAs. This conclusion is based on in vivo analysis of the effect of overproduction of IF-2 on the activity of E. coli tRNA fMet mutants defective in formylation (7, 8) and on in vitro studies of IF-2 interaction with N-blocked aminoacyl-tRNA (9, 10). The formyl group also prevents the tRNA fMet from participating in elongation by blocking binding of elongation factor EF-Tu (5, 7). Formylation of the Met-tRNA fMet is generally accepted as a key checkpoint required for initiation of protein synthesis in eubacteria. This dogma, based primarily on studies conducted in E. coli, was further substantiated by the finding that disruption of the E. coli chromosomal fmt gene severely curtailed cell growth (3). However, some earlier studies have obtained circumstantial evidence that questioned this generalization. A Streptococcus faecalis folate-deficient strain, which is unable to synthesize the formyl donor N 10 -formyltetrahydrofolate (fTHF), was shown to be unaffected in growth or viability (11). However, it was not clear that S. faecalis can initiate protein synthesis in the absence of formylation, since the strain also incurred additional chromosomal mutations that affect proper tRNA modification (11).While the studies in S. faecalis were inconclusive, they hinted that formylation may not be a prerequisite for initiation of protein synthesis in all eubacteria. Therefore, we investig...

Unconventional initiator tRNAs sustain Escherichia coli

“…Even within eubacteria, formylation is not essential, although its deficiency results in a growth compromise (16). In Pseudomonas, efficient initiation occurs with unformylated tRNA with only a moderate growth defect (17). To date, however, the feature of the 3G-C pairs was believed to be indispensable for initiation from both in vitro and in vivo experiments (1,10).…”

Mitochondrial Initiation Factor 2 of Trypanosoma brucei Binds Imported Formylated Elongator-type tRNAMet

“…Initiation of protein synthesis in E. coli depends strictly on formylated tRNA Met-i (32), whereas Pseudomonas aeruginosa is able to use unformylated tRNA (33). Elegant experiments have shown that translation initiation in E. coli can be rendered formylation-independent by complementing an E. coli IF2 mutant with the P. aeruginosa IF2 (34).…”

Determination of the Core of a Minimal Bacterial Gene Set

“…The fmt gene, required for the formylation of methionyl tRNA (EC 2.1.2.9), is also preserved in all the genomes under study, is essential in B. subtilis, and E. coli strains lacking this gene have severely impaired growth rates (30). However, it was not identified in the recently sequenced genome of Phytoplasma asteris (70), and formylation has proven not to be essential for all bacteria, since it is dispensable in Pseudomonas aeruginosa (66), while several E. coli, S. aureus, and S. pneumoniae mutants simultaneously lacking fmt and def (encoding a peptide deformylase) have been obtained (13,55,56,59). Therefore, it has not been included in the minimal gene set.…”