Importance of formylation in eubacterial translation initiation

Abstract

Initiation of protein synthesis in all organisms occurs with the amino acid methionine. In the case of eubacteria and eukaryotic organelles, methionine is further modified to formylmethionine by the enzyme methionyl?tRNA formyltransferase. Formylation is required for efficient channelling of the initiator tRNA into the P?site as it is recognized by the initiation factor IF2 and the formyl group acts as a negative determinant for recognition by the elongation factor EF?Tu. While formylation is clearly essential for translation initiation and normal growth of E. coli, the significance of its role in translation initiation of eubacteria, in general, has been questioned in the past. The aim of the present study is to explore the role of formylation in three eubacterial systems, namely, Pseudomonas aeruginosa, Mycobacterium smegmatis and Escherichia coli. In P. aeruginosa and M. smegmatis, we have successfully established an in vivo initiation assay system that uses a CAT reporter gene with an amber initiation codon and a mutant initiator tRNA with the corresponding CUA anticodon. Using formylation?defective tRNAs for assessing initiation activity, we have shown that formylation does play an important role in the translation initiation of P. aeruginosa. The assay system in M. smegmatis has potential use in understanding translation initiation in the mycobacterial group of organisms. Earlier work has implicated the lack of the ribo?T modification at the 54th position of initiator tRNAfMet_{fMet}fMet? in aiding translation initiation of E. coli in the absence of formylation. The role of this tRNA modification has been further investigated in this study to understand this aspect of formylation?independent initiation.