Importance of formylability and anticodon stem sequence to give a tRNA(Met) an initiator identity in Escherichia coli

Abstract

In bacteria, the free amino group of the methionylated initiator tRNA is specifically modified by the addition of a formyl group. The functional relevance of such a formylation for the initiation of translation is not yet precisely understood. Advantage was taken here of the availability of thefint gene, encoding the Escherichia coli Met-tRNA;Met formyltransferase, to measure the influence of variations in the level of formyltransferase activity on the involvement of various mutant tRNAftet and tRNA:jet species in either initiation or elongation in vivo. The data obtained established that formylation plays a dual role, firstly, by dictating tRNA;Iet to engage in the initiation of translation, and secondly, by preventing the misappropriation of this tRNA by the elongation apparatus. The importance of formylation in the initiator identity of tRNA;Yet was further shown by the demonstration that elongator tRNA~et may be used in initiation and no longer in elongation, provided that it-is mutated into a formylatable species and is given the three G* C base pairs characteristic of the anticodon stem of initiator tRNAs.The recognition of initiator Met-tRNA by specific factors governs the initiation of translation (35). In addition, initiator Met-tRNA must not be recognized by the elongation apparatus, which uses a distinct tRNAMet for the incorporation of internal methionines.In procaryotic cells, prior to its involvement in translation, initiator Met-tRNAr~et is modified by the addition of a formyl moiety on the NH2 group of the esterified methionine. This reaction, catalyzed by 10-formyltetrahydrofolate:L-methionyl-tRNAr et N-formyltransferase (FMT; EC 2.1.2.9), is enough to prevent the incorporation into elongating peptide chains of the methionine esterified to tRNArICI. However, itis not yet known whether formylation is dispensable for Met-tRNAfMet initiator activity. In vitro studies with natural mRNA templates and Escherichia coli extracts showed that the formylation of Met-tRNA;4et enhanced translation rates (5, 12). In particular, the formyl group seemed to improve the efficiency of the selection of tRNAfMet by initiation factor IF2 (10, 24, 34). In vivo, it was found that the initiator activity of glutaminylated tRNAr et variants was related to their formylability (37). Moreover, the characterization of an fint strain pointed out the importance of tRNAP4et formylation in sustaining the rapid growth of E. coli (6).Besides determinants involved in its formylation, tRNAr et may carry other structural determinants involved in its functional specificity towards the initiation step. consisted of an in vivo assay in which variants of initiator or elongator tRNAs were exposed to various intracellular levels of FMT activity. The variants of tRNAfMe; and tRNA~et were constructed on the basis of the precise knowledge of the nucleotides governing the recognition of Met-tRNA1fet

Initiator transfer RNAs

“…These studies confirm the results of in vitro analyses and show that all the mutants that have a 1:72 base pair are active in elongation in vivo in E. coli and in Saccharomyces cerevisiae (10,19,29,35).…”

“…This explains how a relatively limited number of nucleotides in the tRNA can specify all of the properties needed for initiation of protein synthesis. Further confirmation of this comes from the finding that introduction of the minimal sequences necessary for formylation and for P site binding onto elongator methionine and glutamine tRNAs allows these mutant tRNAs to act as initiator tRNAs in E. coli (10,34).…”

The Pseudomonas aeruginosa Initiation Factor IF-2 Is Responsible for Formylation-independent Protein Initiation in P. aeruginosa

“…1 (1)(2)(3)(4)(5)(6). In vitro studies of protein synthesis using extracts from Escherichia coli showed that translation was stimulated by fMettRNA fMet (7).…”

Evidence that peptide deformylase and methionyl-tRNA(fMet) formyltransferase are encoded within the same operon in Escherichia coli

“…The product of the fint gene plays an essential role for optimal growth of E. coli cells (13), possibly by enabling MetMet tRNAf to participate in the initiation rather than in the elongation step of the translation process (12). fnt is the second cistron of an operon whose first cistron, which we called fins, encodes a putative 19-kDa polypeptide (12,21). The translated amino acid sequence of fins shows a motif characteristic of neutral zinc aminopeptidases (16,17,25,29) (Fig.…”

“…However, peptide deformylase could not be further characterized because of its extreme instability (1,18,28 (13). The product of the fint gene plays an essential role for optimal growth of E. coli cells (13), possibly by enabling MetMet tRNAf to participate in the initiation rather than in the elongation step of the translation process (12). fnt is the second cistron of an operon whose first cistron, which we called fins, encodes a putative 19-kDa polypeptide (12,21).…”