1. Introduction
The cellular protein synthesis, a key process in gene expression, is orchestrated by multiple players including ribosomes, mRNAs, tRNAs and the translation factors. Bacterial ribosomes synthesize proteins in four distinct steps of initiation, elongation, termination, and ribosome recycling (Fig. 1 ) using a distinct set of translation factors for each step (Gold 1988; Laursen et al. 2005). The initiation step uses three initiation factors, IF1, IF2 and IF3 and employs, almost exclusively, a special tRNA called initiator tRNA (tRNAfMet or i-tRNA) (Gualerzi et al. 2014; Gualerzi and Pon 1990). In all organisms, i-tRNA is charged with methionine (Met-i-tRNA) by methionyl-tRNA synthetase (Meinnel et al. 1990). In bacteria and eukaryotic organelles (mitochondria and chloroplasts), the Met-i-tRNA is then formylated by formylmethionine transferase (Fmt) to fMet-i-tRNA (also referred to simply as i-tRNA) using N10-formyltetrahydrofolate (N10-fTHF) as the formyl group donor (Kozak 1983; Shetty and Varshney 2021). Thus, i-tRNA mediated initiation with Met (or fMet), irrespective of the nature of the initiation codon, is also conserved in all domains of life (Kozak 1983). The initiation step is meticulously regulated (Gold 1988; Gualerzi and Pon 1990). It not only ensures conservation of cellular energy but also prevents toxicity that may result from accumulation of faulty proteins in the cell. The canonical pathway of initiation, a major mechanism in bacteria (Höfig et al. 2019), begins with the binding of i-tRNA and mRNA on 30S subunit (30S) in the presence of the three initiation factors by forming a 30S pre-initiation complex (30S PIC). The 30S PIC then rearranges to 30S IC to allow accurate recognition of initiation codon in the mRNA by i-tRNA anticodon in the P-site. A 70S complex, competent to transit into the elongation step, is then formed by joining of 50S ribosomal subunit (50S) and release of the initiation factors. The initiation factors play essential roles in initiation. IF1 facilitates the functions of IF2 and IF3, and its 30S binding blocks the A-site. IF2, because of its affinity to the formyl group in fMet-i-tRNA facilitates its localization to the P-site. Also, as IF2 has affinity to 50S (Boileau et al. 1983; Heimark et al. 1976; La Teana, Gualerzi, and Dahlberg 2001), it helps its docking to 30S IC. IF3, on the other hand, increases off-rates of tRNAs that bind to 30S (Antoun et al. 2006a; Haggerty and Lovett 1997; Hartz et al. 1990; Hartz, McPheeters, and Gold 1989; Meinnel et al. 1999; Sussman, Simons, and Simons 1996; Tedin et al. 1999) and also serves as an anti-association factor, which allows proper assembly of 30S IC prior to joining of 50S (Dallas and Noller 2001; Godefroy-Colburn et al. 1975; Karimi et al. 1999; Sacerdot et al. 1996). The rate at which 50S joins 30S IC is crucial for accuracy of initiation. Faster rates of subunit docking may lead to inaccurate initiation because of trapping incorrect tRNA at the P-site (Antoun et al. 2006b, 2006a). The selection of i-tRNA at the P-site is a regulated process, and various ribosomal elements add to the fidelity of i-tRNA binding. For example, 16S rRNA bases A1339 and G1338 monitor the G29-C41 and G30-C40 base pairs (the first two GC pairs) by type-I and type-II A-minor interactions, respectively (Selmer et al. 2006). Methylations of G966 and C967 to m2G966 and m5C967 by RsmD and RsmB, respectively, contribute to the interactions between i-tRNA anticodon and the initiation codon in the mRNA (Arora, Bhamidimarri, Weber, et al. 2013; Selmer et al. 2006; Seshadri et al. 2009). Likewise, methylations of A1518 and A1519 to m6A1518 and m6A1519, respectively in 16S rRNA by RsmA enhance the fidelity of i-tRNA binding (Demirci et al. 2010). The modifications at A1518 and A1519 are also important for 30S biogenesis (Connolly, Rife, and Culver 2008; Demirci et al. 2010). In addition, the C-terminal tails of ribosomal proteins (r-proteins), uS9 and uS13 that extend into the P-site, contribute to i-tRNA selection (Arora, Bhamidimarri, Weber, et al. 2013). The r-protein, uS12, despite being a resident of the A-site contributes to i-tRNA selection and the fidelity of translation initiation (Datta, Pillai, et al. 2021; Datta, Singh, et al. 2021; Hoang, Fredrick, and Noller 2004; Selmer et al. 2006).
Once 70S complex is formed, it is competent to transit into the elongation step to translate the remainder of the open reading frame (ORF) in the mRNA by recruiting aminoacyl-tRNAs (in complex with elongation factor Tu, EFTu) in the ribosomal A-site (A-site) as directed by the ORF. Accommodation of aminoacyl-tRNA in the A-site triggers peptide bond formation between the amino acid it (A-site tRNA) carries and the peptide (or formyl-amino acid) the P-site tRNA harbours, positioning the two tRNAs in the P/E and A/P hybrid states. The hybrid state of the ribosome so formed is brought back to the classical state by the translocation step facilitated by elongation factor G (EFG) to accommodate the next codon in the A-site. The elongation cycles repeat until a termination codon is positioned in the A-site for its recognition by a release factor (RF1 or RF2) to release the peptide chain from the P-site peptidyl-tRNA. The RF1/RF2 are recycled by RF3 (Freistroffer et al. 1997) followed by recycling of the post-termination complex (ribosome, mRNA, tRNA) by the concerted action of ribosome recycling factor (RRF), EFG and IF3 to enable a new cycle of initiation (Borg, Pavlov, and Ehrenberg 2016; Prabhakar et al. 2017). Despite cells having evolved with diverse mechanisms to uphold the accuracy and efficiency of different translation steps, the translating ribosomes often encounter stalls during elongation. The stalled ribosomes are recycled by tmRNA (SsrA) mediated trans-translation (Keiler, Waller, and Sauer 1996), alternate ribosome rescue mechanisms that use ArfA and ArfB (Abo and Chadani 2014) or other less understood processes that release (drop-off) peptidyl-tRNAs from ribosomes. Accumulation of peptidyl-tRNAs in cell is toxic as it sequesters tRNAs and makes them unavailable for the elongation step. The cells avoid accumulation of peptidyl-tRNAs by possessing peptidyl-tRNA hydrolase (Pth), which cleaves the ester bond between the peptide and tRNA (Menninger 1978, 1979; Menninger et al. 1983; Singh et al. 2005, 2008; Singh and Varshney 2004).
The details of the various steps in protein synthesis and the translation apparatus outlined above have been extensively investigated by genetic, biochemical, and structural approaches, to unravel the fundamental mechanisms since the history of molecular biology. In addition, the three-dimensional structural determinations of ribosomes providing snapshots of the translational events in atomic details, in the relatively more recent times, have remarkably advanced our understanding of the ribosome function (Green and Noller 1997; Jobe et al. 2019; Korostelev 2022; Noller 2005; Noller et al. 2017; Noller, Donohue, and Gutell 2022; Ramakrishnan 2002, 2011; Samhita and Varshney 2010; Schmeing and Ramakrishnan 2009; Voorhees and Ramakrishnan 2013). The role of changes in the ribosomal composition (ribosomal heterogeneity) has also been implicated in differential translation of mRNAs (Byrgazov, Vesper, and Moll 2013; Genuth and Barna 2018; Shi et al. 2017; Zhang et al. 2022). However, much remains to be probed to understand the intricacies of the ribosome function, regulation, and the evolution of the translation apparatus to respond to the changes in the cellular physiology/metabolic state.
We have investigated the regulation of translation initiation in bacteria, using molecular genetics approaches employing analysis ofE. coli suppressors which allow initiation with a mutant i-tRNA defective for its participation in initiation. The findings have allowed us to address the evolutionary significance of the special features of i-tRNA and its interactions with the translation apparatus including the ribosome biogenesis factors. Further, the studies have revealed that execution of faithful translation initiation depends critically on one-carbon metabolism. However, to elaborate on these findings, it is important to introduce the special features of i-tRNA and the design of the in vivo assay system used in the suppressor analysis.