5. Conclusions and perspectives
The use of Met or fMet for initiation connects protein synthesis with OCM, whereby an energy rich state (higher flux of Met and N10-fTHF) of the cell would favour translation initiation, and an energy depleted state (lower flux of Met and N10-fTHF) would downregulate initiation. Such a regulation averts the cell from undertaking the highly energy expensive process of protein synthesis under energy deficient conditions and avoids accumulation of incomplete proteins and, toxicity to the cell. On an application front, the connection between translation initiation and OCM offers improved ways of inhibiting bacterial growth. The FolD mutant strains are hypersensitive to further perturbation of OCM by TMP (inhibitor of dihydrofolate reductase) (Lahry et al. 2020) suggesting that augmentation of the age-old sulfa drugs (which impact production of DHF by targeting dihydropteroate synthase) with FolD inhibitors could be an important antibacterial strategy.
In bacteria and the eukaryotic organelles, formylation of i-tRNA directs it to the initiation step and prevents its binding to EFTu. The lack of formylation of i-tRNA in real time leads to its binding to EFTu and its participation at the step of elongation (Shah et al. 2019). These observations allow better understanding of how a single tRNAMet (with features of i-tRNA) participates at the steps of initiation and elongation in mammalian mitochondria (Govindan et al. 2018). In eukaryotes (cytosol), the presence of A1:U72, and the structural features in TψC arm of mammalian i-tRNA, and the 2′-O-phosphoribosyl modification at position 64 in yeast i-tRNA avoid i-tRNA binding to eEF1A (Desgrès et al. 1989; Drabkin, Estrella, and Rajbhandary 1998). Together with the manner in which i-tRNA is delivered to the P-site in eukaryotes (or in archaea), allows them to do away with the requirement of formylation (Benelli and Londei 2011; Jackson, Hellen, and Pestova 2010). In fact, formylation of i-tRNA in eukaryotes is detrimental (Kim et al. 2018; Ramesh, Köhrer, and RajBhandary 2002). Separately, our studies (Shah et al. 2019; Shetty et al. 2016; Shetty and Varshney 2016) and those of others (Nilsson et al. 2006) showed that the lack of formylation in bacteria can be rescued by increased abundance of i-tRNA in cell. Thus, together with the observations in eukaryotes, it may well be that in bacteria, formylation may have no additional functions beyond the initial recruitment of i-tRNA to the ribosomes.
However, the feature of the 3GC pairs in the anticodon stem, highly conserved in all i-tRNAs, not only facilitates i-tRNA binding to ribosome but also helps in stabilizing its interactions in the P-site during the various stages of initiation that convert 30S PIC to 70S complex competent to transit to elongation step. Importantly, the 3GC pairs are also crucial in the release of IF3 from the 70S complex (Shetty et al. 2017) and in the final maturation of 17S rRNA to 16S rRNA (Shetty and Varshney 2016). However, at least in a reporter system, the strict requirement of the 3GC pairs is functionally compensated for by an extended interaction between the SD and aSD sequences (Shetty et al. 2014). The G1338 and A1339, which establish A-minor interactions with the 3GC pairs (Lancaster and Noller 2005; Selmer et al. 2006) and the methylations of 16S rRNA nucleosides (Arora, Bhamidimarri, Bhattacharyya, et al. 2013; Das et al. 2008; Seshadri et al. 2009) are responsible for the functions of the 3GC pairs. Nonetheless, for a better understanding of the various roles of the 3GC pairs, knowledge of the dynamics of these interactions (or the network of interactions) is essential.
The bacteriophages producing ribonuclease toxins targeting i-tRNA, or the stress/starvation conditions, may deplete i-tRNA levels in bacteria. The deficiency of i-tRNA impacts ribosome maturation. Nutritional deficiency may also limit S-adenosyl-methionine (SAM) levels impacting methylations in rRNA and r-proteins, leading to heterogeneity in the ribosomes, which may influence proteome diversity (for example, by initiation with elongator tRNA) (Fig. 8) . However, a direct connection between the levels of i-tRNA or the heterogeneity of methylations of the rRNA nucleosides, and the changes in proteome has not been made possibly because the changes are subtle.
The role of RluD in 30S maturation was serendipitous, and the precise mechanism of how it releases RbfA from 30S remains unknown. RluD may do so by interacting directly with the 30S or that its binding to H69 (50S) may affect RbfA release during its (50S) docking onto the 30S in the pioneering round of initiation. The latter is explicable by the structure of the RluD docked 50S subunit (Vaidyanathan, Deutscher, and Malhotra 2007). In this model, the C-terminal tail of RluD protruding from the 50S would contact the 30S subunit to release RbfA before actual subunit joining. Further, biochemical and structural studies are required to explore the detailed molecular mechanisms of the role of RbfA in lowering the fidelity of initiation, and for the role of RluD (or IF3) in RbfA release from 30S.
Finally, an area of research where our understanding is inadequate, is the role of RRF in the fidelity of initiation. Genetic studies have clearly shown a connection between RRF, uS12, IF3 and Pth (Das and Varshney 2006; Datta, Pillai, et al. 2021; Singh and Varshney 2004). Interestingly, role of RRF in fidelity of initiation provides with a novel mechanism to not only ensure correct assembly of 70S complexes but also in the scrutiny of its transition into the elongation step by acting on the early-stage elongation complexes. Though the events of translation initiation are well characterized, this review documents novel findings that have furthered our understanding of the intricacies of faithful translation initiation and highlights the caveats that could be explored in future for a comprehensive understanding of the same.