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.