2. Special features of i-tRNA
Organisms possess two distinct methionine tRNAs, the i-tRNA and elongator tRNAMet. Both the tRNAs possess CAU anticodons. The i-tRNA decodes the initiation/start codons (AUG or a related codon), and elongator tRNAMet decodes the AUG codons found at internal positions in ORF. Like other tRNAs, these tRNAs possess typical clover-leaf secondary structures. However, i-tRNAs harbour at least three distinct structural features that distinguish them from the elongator tRNAs (Fig. 2A ). Firstly, there exists a Watson-Crick mismatch between nucleotides 1 and 72 (C1x A72 in E. coli ) at the top of the acceptor stem (in contrast to the Watson-Crick base pairs found in elongator tRNAs at this position). The 1x72 pair mismatch is crucial for, (i) recognition of i-tRNA by Fmt (along with G2-C71, C3-G70, and A11-U24 base pairs) for formylation of the amino acid attached to it; (ii) prevention of fMet-i-tRNA (a form of peptidyl-tRNA) from hydrolysis by peptidyl-tRNA hydrolase (Pth), whose role is to recycle tRNAs from peptidyl-tRNAs (Das and Varshney 2006); and (iii) avoidance of its binding to EFTu and the ambiguity of its participation at the step of elongation (Dutka et al. 1993; Guillon et al. 1992; Hansen et al. 1986; Lee et al. 1992; Lee, Seong, and RajBhandary 1991; Ramesh et al. 1997; Schulman and Pelka 1975; Seong, Lee, and RajBhandary 1989; Seong and RajBhandary 1987; Varshney et al. 1991). Secondly, the bacterial i-tRNAs possess a Pu11-Py24 base pair (unlike Py11-Pu24 base pair in elongator tRNAs). This position has been shown to impact formylation of the amino acid attached to i-tRNA (Lee et al. 1991). Thirdly, all i-tRNAs possess a highly conserved feature of three consecutive GC base pairs (G29-C41, G30-C40, G31-C39, also stated as GC/GC/GC or 3GC pairs) in the anticodon stem (Fig. 2A, i ), which enable its direct binding to the P-site (Mandal et al. 1996; Marck and Grosjean 2002; Mayer et al. 2001; Varshney, Lee, and RajBhandary 1993). These special features of i-tRNA not only distinguish it from elongator tRNAMet (Fig. 2A) but also, along with IFs, allow it to outcompete elongator tRNAs from binding into the P-site. While the 3GC pairs in i-tRNA anticodon stem are highly conserved in all domains of life, the first and third GC pairs (G29-C41, and G31-C39) have been found to be replaced by A-U or G-U pairs in organisms such as mycoplasma, and α-proteobacterial species (Ayyub et al. 2018; Dong et al. 2014; Samhita, Shetty, and Varshney 2012). The middle one (G30-C40) is the most crucial GC pair in the bacterial i-tRNAs. In addition, it was shown that the first and the third GC pairs are dispensable even in E. coli (Samhita et al. 2012). In fact, even the second G-C pair could be changed to a G-U pair in some sequence contexts (Samhita et al. 2012; Shetty et al. 2017). Although it should also be said that the mutations in the 3GC pairs do not support a healthy growth of E. coli (Shetty et al. 2017).