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\begin{document}

\title{Fidelity of translation initiation in bacteria: an initiator
tRNA-centric view}



\author[1]{Umesh Varshney}%
\author[1]{Kuldeep Lahry}%
\author[1]{Madhurima Datta}%
\affil[1]{Indian Institute of Science Department of Microbiology and Cell Biology}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Translation of messenger RNA (mRNA) in bacteria occurs in the steps of
initiation, elongation, termination and ribosome recycling. The
initiation step comprises multiple stages and uses a special transfer
RNA (tRNA) called initiator tRNA (i-tRNA), which is first aminoacylated
and then formylated using methionine and N
\textsuperscript{10}-formyl-tetrahydrofolate (N
\textsuperscript{10}-fTHF), respectively. Both methionine and N
\textsuperscript{10}-fTHF are produced via one-carbon metabolism,
linking translation initiation with active cellular metabolism. The
fidelity of i-tRNA binding to the ribosomal peptidyl-site (P-site) is
attributed to the structural features in its acceptor stem, and the
highly conserved three consecutive G-C base pairs (3GC pairs) in the
anticodon stem. While the acceptor stem region is important in
formylation of the amino acid attached to i-tRNA and its initial binding
to the P-site, the 3GC pairs are crucial in transiting i-tRNA through
various stages of initiation. We utilized the feature of 3GC pairs to
investigate the nuanced layers of scrutiny that ensure fidelity of
translation initiation through i-tRNA abundance and its interactions
with the components of the translation apparatus. We discuss the
importance of i-tRNA in the final stages of ribosome maturation, as also
the roles of the Shine-Dalgarno sequence, ribosome heterogeneity,
initiation factors, ribosome recycling factor and coevolution of the
translation apparatus in orchestrating a delicate balance between the
fidelity of initiation and/or its leakiness to generate proteome
plasticity in cells to confer growth fitness advantages in response to
the dynamic nutritional states.%
\end{abstract}%



\sloppy


\textbf{Fidelity of translation initiation in bacteria: an initiator
tRNA-centric view}

Kuldeep Lahry\textsuperscript{1\#}, Madhurima
Datta\textsuperscript{1+}and Umesh Varshney\textsuperscript{1,2*}

\textsuperscript{1}Department of Microbiology and Cell Biology, Indian
Institute of Science, Bengaluru 560012

\textsuperscript{2}Jawaharlal Nehru Centre for Advanced Scientific
Research, Jakkur, Bengaluru 560064

*\emph{Correspondence to} : Umesh Varshney, Phone: +918022932686, Fax:
+918023602697,

E-mail: varshney@iisc.ac.in; uvarshney@gmail.com

\textsuperscript{\#}Current address: IGF, Univ. Montpellier, CNRS,
INSERM, Montpellier, France

\textsuperscript{+}Current address: Advanced Centre for Treatment,
Research and Education in Cancer, TMC, Navi Mumbai, India 410210.

\textbf{Keywords:} Ribosome maturation, 3GC base pairs, Shine-Dalgarno
sequence, one-carbon metabolism, RluD, FolD, formylation,
10-formyltetrahydrofolate, rRNA methylations, ribosome recycling, RRF,
initiation factors



\textbf{Abstract:}

Translation of messenger RNA (mRNA) in bacteria occurs in the steps of
initiation, elongation, termination and ribosome recycling. The
initiation step comprises multiple stages and uses a special transfer
RNA (tRNA) called initiator tRNA (i-tRNA), which is first aminoacylated
and then formylated using methionine and
N\textsuperscript{10}-formyl-tetrahydrofolate
(N\textsuperscript{10}-fTHF), respectively. Both methionine and
N\textsuperscript{10}-fTHF are produced via one-carbon metabolism,
linking translation initiation with active cellular metabolism. The
fidelity of i-tRNA binding to the ribosomal peptidyl-site (P-site) is
attributed to the structural features in its acceptor stem, and the
highly conserved three consecutive G-C base pairs (3GC pairs) in the
anticodon stem. While the acceptor stem region is important in
formylation of the amino acid attached to i-tRNA and its initial binding
to the P-site, the 3GC pairs are crucial in transiting i-tRNA through
various stages of initiation. We utilized the feature of 3GC pairs to
investigate the nuanced layers of scrutiny that ensure fidelity of
translation initiation through i-tRNA abundance and its interactions
with the components of the translation apparatus. We discuss the
importance of i-tRNA in the final stages of ribosome maturation, as also
the roles of the Shine-Dalgarno sequence, ribosome heterogeneity,
initiation factors, ribosome recycling factor and coevolution of the
translation apparatus in orchestrating a delicate balance between the
fidelity of initiation and/or its leakiness to generate proteome
plasticity in cells to confer growth fitness advantages in response to
the dynamic nutritional states.



\textbf{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 (\textbf{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
(tRNA\textsuperscript{fMet} 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
N\textsuperscript{10}-formyltetrahydrofolate
(N\textsuperscript{10}-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 (H\selectlanguage{ngerman}öfig 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 G\textsubscript{29}-C\textsubscript{41} and
G\textsubscript{30}-C\textsubscript{40} 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 m\textsuperscript{2}G966
and m\textsuperscript{5}C967 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
m\textsuperscript{6}A1518 and m\textsuperscript{6}A1519, 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
of\emph{E. 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 \emph{in vivo} assay system used in the suppressor analysis.

\textbf{2. Special features of i-tRNA}

Organisms possess two distinct methionine tRNAs, the i-tRNA and
elongator tRNA\textsuperscript{Met}. Both the tRNAs possess CAU
anticodons. The i-tRNA decodes the initiation/start codons (AUG or a
related codon), and elongator tRNA\textsuperscript{Met} 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 (\textbf{Fig. 2A} ). Firstly, there exists
a Watson-Crick mismatch between nucleotides 1 and 72
(C\textsubscript{1}x A\textsubscript{72} in \emph{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
G\textsubscript{2}-C\textsubscript{71},
C\textsubscript{3}-G\textsubscript{70}, and
A\textsubscript{11}-U\textsubscript{24} 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
Pu\textsubscript{11}-Py\textsubscript{24} base pair (unlike
Py\textsubscript{11}-Pu\textsubscript{24} 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
(G\textsubscript{29}-C\textsubscript{41},
G\textsubscript{30}-C\textsubscript{40},
G\textsubscript{31}-C\textsubscript{39}, also stated as GC/GC/GC or 3GC
pairs) in the anticodon stem (\textbf{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
tRNA\textsuperscript{Met} \textbf{(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
(G\textsubscript{29}-C\textsubscript{41}, and
G\textsubscript{31}-C\textsubscript{39}) have been found to be replaced
by A-U or G-U pairs in organisms such as mycoplasma, and
\selectlanguage{greek}α-\selectlanguage{english}proteobacterial species (Ayyub et al. 2018; Dong et al. 2014; Samhita,
Shetty, and Varshney 2012). The middle one
(G\textsubscript{30}-C\textsubscript{40}) 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 \emph{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 \emph{E. coli} (Shetty et al. 2017).

\textbf{3. \emph{In vivo} assay system for genetic analysis of
initiation}

To develop a plasmid based assay system for \emph{in vivo} analyses of
the structure function relationship of i-tRNA, chloramphenicol (Cm)
acetyltransferase (CAT) gene was mutated to encode
CAT\textsubscript{am1} mRNA possessing UAG as initiation codon (in place
of AUG) along with two other mutations at codons 2 and 5 to avoid
background initiation (Fig. 2B, i) (Varshney and RajBhandary 1990). A
corresponding change was also made in i-tRNA gene (\emph{metY} ) to
encode an i-tRNA with CUA anticodon (i-tRNA\textsubscript{CUA}) to allow
it to pair with the UAG initiation codon in CAT\textsubscript{am1}
mRNA.\emph{E. coli} harbouring CAT\textsubscript{am1} gene (on a
plasmid, pCAT\textsubscript{am1}) gained resistance to Cm
(Cm\textsuperscript{R}) only if it also simultaneously possessed i-tRNA
gene (\emph{metY} \textsubscript{CUA}) to encode
i-tRNA\textsubscript{CUA}. The pCAT\textsubscript{am1}\emph{metY}
\textsubscript{CUA} (harbouring both the CAT\textsubscript{am1} and
\emph{metY} \textsubscript{CUA}genes) provided an assay system for
\emph{in vivo} initiation (\textbf{Fig. 2B, i and ii} ). Any mutants of
i-tRNA\textsubscript{CUA}that continue to confer Cm\textsuperscript{R}
to \emph{E. coli} , are functional as initiators; and those that are Cm
sensitive (Cm\textsuperscript{S}) are non-functional as initiators. In
this assay system, initiation occurred predominantly with fGln rather
than with fMet (the CAU to CUA change in the anticodon, the mutant
i-tRNA\textsubscript{CUA} becomes highly deficient for recognition by
MetRS but it becomes a good substrate for GlnRS (Schulman and Pelka
1985; Seong et al. 1989)). Gln attached to i-tRNA is also an excellent
substrate for Fmt, an enzyme that formylates the \selectlanguage{greek}α-\selectlanguage{english}amino group of most
amino acids attached to i-tRNA (Mayer et al. 2003). Importantly, the
assay system showed that even though Met is evolutionarily conserved as
an initiating amino acid, initiation can occur with other amino acids
even \emph{in vivo} . Further, the assay system showed that when the
mismatch at 1-72 position is converted to a Watson-Crick pair, the
mutant i-tRNA becomes a dual function tRNA that participates both at the
initiation and elongation steps (Govindan, Ayyub, and Varshney 2018;
Varshney et al. 1991). When the special features of i-tRNA found in its
acceptor stem and anticodon stem were transplanted into elongator tRNAs,
they gained initiator function (Varshney et al. 1993). Not surprisingly,
when the i-tRNA 3GC pairs in the anticodon stem were changed with those
found in the elongator
tRNA\textsuperscript{Met}(U\textsubscript{29}-A\textsubscript{41},
C\textsubscript{30}-G\textsubscript{40},
A\textsubscript{31}-?\selectlanguage{english}\textsubscript{39} or 3GC mutant i-tRNA or
ua/cg/au; mutations shown in small letters), the i-tRNA mutant failed to
function in initiation \textbf{(Fig. 2B, iii)} despite its efficient
aminoacylation and formylation (Das et al. 2008; Mandal et al. 1996;
Shah et al. 2019).

\textbf{4. Regulation of the fidelity of translation initiation and
ribosome biogenesis}

The failure of the 3GC mutant i-tRNA in initiation \emph{in vivo} ,
formed the basis for our genetic screen to isolate suppressor mutations
(in the genome) that allow initiation with the 3GC mutant i-tRNA
(\textbf{Fig. 2B, iv} ). A detailed characterisation and molecular
analysis revealed the roles of, (i) i-tRNA gene copy number and its
abundance; (ii) one-carbon metabolism, Fmt and FolD; and (iii) ribosomal
components, ribosome biogenesis factors, translation factors and their
coevolution; in the fidelity of translation initiation. In addition, the
suppressors have provided newer insights into the process of ribosome
biogenesis/maturation. We discuss these findings in the context of
translation initiation, and ribosome biogenesis.

\textbf{(i) Role of i-tRNA in translation initiation and ribosome
biogenesis.}

\emph{\textbf{(a) Initiator tRNA gene copy numbers and its abundance in
the fidelity of initiation:} E. coli} possesses four copies of i-tRNA
genes. Of these, three are located at 63.5' as \emph{metZWV} operon
(tRNA\textsuperscript{fMet1}); and the fourth one,
\emph{metY}(tRNA\textsuperscript{fMet2}) is located at 71.5' (Kenri,
Imamoto, and Kano 1994). All four copies of i-tRNAs in \emph{E. coli}
B-strains are identical (Mandal and RajBhandary 1992). However, in the
K-strains, tRNA\textsuperscript{fMet2} harbours an A at position 46,
whereas other i-tRNAs (tRNA\textsuperscript{fMet1}) harbour
m\textsuperscript{7}G at this position
(m\textsuperscript{7}G\textsubscript{46}). The significance of retention
of i-tRNAs with m\textsuperscript{7}G\textsubscript{46} and
A\textsubscript{46} in\emph{E. coli} K strains is unclear. Nonetheless,
either of the loci is sufficient for survival of \emph{E. coli} (Kenri,
Imamoto, and Kano 1992; Tsuyoshi et al. 1991). The cellular abundance of
i-tRNA is proportional to the copy number of i-tRNA genes (Kapoor, Das,
and Varshney 2011). Deletion of \emph{metZWV} reduces \emph{E. coli}
growth and confers cold sensitivity phenotype to it. However, deletion
of\emph{metY} has almost no effect on the growth of the strain. Samhita
and colleagues found that \emph{E. coli} strains with four i-tRNA genes
outcompeted a strain with three i-tRNA genes when grown in rich medium.
However, the opposite was true when they were grown in nutrient-poor
conditions for a prolonged time (Samhita, Nanjundiah, and Varshney
2014). These observations suggested that i-tRNA genes, in bacteria, may
be subjected to dynamic copy number changes. The presence of \emph{E.
coli} strains with five i-tRNA genes (HS) was reported in the gut
(nutrient-rich) and with three i-tRNA genes (IAI39) was identified in
urinary tract infections (nutrient-poor). In our genetic analyses, a
severe depletion of i-tRNA in \emph{E. coli} either by promoter down
mutations (as present in a series of suppressor strains identified in
our genetic screen) or by engineering a deletion of the
entire\emph{metZWV} locus (Kapoor et al. 2011), revealed a link between
i-tRNA abundance and the fidelity of initiation. Depletion of cellular
i-tRNA allowed initiation not only with the 3GC mutant i-tRNA and the
noncanonical i-tRNAs lacking a full complement of 3GC pairs in the
anticodon stem, but also with elongator tRNAs (\textbf{Fig. 3, ii} )
(Kapoor et al. 2011; Samhita et al. 2013). Thus, a high abundance of
canonical i-tRNAs is required not only to overcome the rate-limiting
step of translation initiation (Gualerzi and Pon 1990; Laursen et al.
2005) but also to discriminate against binding of ''i-tRNA like'' or
elongator tRNAs in the P-site (Kapoor et al. 2011; Samhita et al. 2013).
These observations allowed us to then engineer the growth of \emph{E.
coli} exclusively on i-tRNAs lacking either the first GC pair, the third
GC pair or both the first and the third GC pairs as found in some
mycoplasma and rhizobium species (Samhita et al. 2012). In a recent
study where all the components of the translation machinery and entire
translatome of the human pathogen \emph{Mycoplasma
pneumoniae}(\emph{Mpn} ) were analysed, the abundance of i-tRNAs was
found to be massive at 12.1\% as opposed to \textasciitilde{}3\% in
\emph{E. coli}(Dong, Nilsson, and Kurland 1996; Weber et al. 2023).
Given that the anticodon stem of i-tRNA in \emph{Mpn} harbours AU/GC/GU
sequence instead of the canonical GC/GC/GC, the high abundance of i-tRNA
could be the organism's way of outcompeting ``i-tRNA like'' or elongator
tRNA binding in the P-site, mitigating loss of initiation fidelity.

These observations raise a question if there are any natural means of
regulating fidelity of translation initiation by regulating i-tRNA
abundance in bacteria? Several lines of evidence suggest that \emph{E.
coli} could regulate the levels of its i-tRNA contents depending on the
nutritional status. At least \emph{in vitro} , ppGpp has been shown to
downregulate expression of \emph{metZWV} during the stringent response
(Takahiro, Shunsuke, and Fumio 1988). Likewise, expression
from\emph{metY} may be regulated by cAMP-CAP a global regulator of
transcription, as well as ArgR, a specific transcriptional regulator of
arginine metabolism (Krin et al. 2003). Moreover, studies have shown
that if \emph{E. coli} is deprived of leucine, the level of
aminoacylated i-tRNAs decreases dramatically (Dittmar et al. 2005).
Another mechanism of downregulating i-tRNA levels is based on the action
of VapC toxin of the VapBC toxin-antitoxin module in \emph{Shigella
flexneri} 2a and the VapCLT2 of Salmonella enterica serovar Typhimurium
LT2, both of which are site-specific tRNases that target 3GC pairs in
the i-tRNA anticodon stem (Winther and Gerdes 2011). Based on our
findings, such a depletion of i-tRNA will favour initiation with
unconventional i-tRNAs or elongator tRNAs. In yeast, i-tRNA depletion
prompts translation of GCN4, a nutritional stress transcription factor
(Conesa et al. 2005). Further, the levels of initiator and elongator
methionine tRNAs are negatively associated with cell proliferation
versus quiescence (Kanduc 1997). Thus, it seems that downregulating
i-tRNA levels could be a cellular response to overcome stress by
promoting `leakiness' in the translation apparatus.

(b) Initiator tRNA abundance and ribosome maturation: Another
consequence of i-tRNA depletion we observed in \emph{E. coli}was the
gain of cold sensitive phenotype in the strains deleted for\emph{metZWV}
. We showed that the i-tRNA, more specifically its 3GC pairs, play a
crucial role in the terminal stages of ribosome maturation by prompting
trimming of the extra sequences at the 3' and 5' ends of the 17S
precursor to produce mature 16S rRNA during the pioneering round of
initiation \textbf{(Fig. 4)} . Based on the genetic interactions, the
extra sequences may be trimmed by RNase R, RNase II, and RNase PH
(Samhita et al. 2012; Shetty and Varshney 2016; Tsuyoshi et al. 1991).
More recently, based on the analysis of lamotrigine toxicity (which
targets IF2), we showed that this role of i-tRNA in maturation of 16S
rRNA is mediated through IF2 and i-tRNA complex bound to the 30S. Also,
lamotrigine mediated inhibition of ribosome biogenesis led to an
increased accumulation of ribosome binding factor A (RbfA), a late stage
ribosome biogenesis factor, on 30S (Singh et al. 2023).

In other investigations, we noted that overexpression of i-tRNA rescues
biogenesis defects resulting from the deficiency of methylations at G966
and C967 in 16S rRNA in the P-site or deletion of the C-terminal
residues of uS9 (S126, K127 and R128, or SKR) that impact i-tRNA binding
(Arora, Bhamidimarri, Bhattacharyya, et al. 2013; Ayyub et al. 2018).
The binding of i-tRNA may affect the 3' end region of 16S rRNA through
conserved residues, for example, G1338 and A1339, which interact with
the 3GC pairs for the accuracy of i-tRNA selection in initiation. The
conformational changes induced in the ribosome during initiation might
then signal the final processing of the 17S rRNA to 16S rRNA.

\textbf{(ii) Role of one-carbon metabolism, Fmt and FolD in the fidelity
of translation initiation.}

One-carbon metabolism (OCM), a central pathway for synthesis of many
amino acids, nucleotides, and folate species comprising one-carbon unit
intermediates (formyl, methenyl, methyl, and methylene) impacts the rate
and fidelity of translation in ways that include availability of amino
acids and modifications of translation factors, tRNAs, rRNAs, mRNAs, and
r-proteins. A detailed review of translational regulation by OCM was
recently published~(Shetty and Varshney 2021). Formylation of i-tRNA
increases its affinity towards IF2, and contributes to its selection in
the P-site in bacteria, mitochondria and chloroplasts (Majumdar et al.
1976; Sundari et al. 1976). Formylation of i-tRNA is carried out by Fmt,
and a formyl group donor, N\textsuperscript{10}-fTHF (\textbf{Fig. 3, i}
). Interestingly, Fmt can also utilize N\textsuperscript{10}-formyl
dihydrofolate (N\textsuperscript{10}-fDHF) as a poor alternate for
N\textsuperscript{10}-fTHF (Sah and Varshney 2023). The deletion
of\emph{fmt} (gene encoding Fmt) causes modest to severe growth defects
in\emph{Pseudomonas aeruginosa} and \emph{E. coli} , respectively
(Guillon et al. 1992; Newton, Creuzenet, and Mangroo 1999) and is not
essential for survival of these bacteria. However, as we discuss in the
following paragraphs, formylation of i-tRNA increases efficiency and
accuracy of initiation (Lahry et al. 2020; Shah et al. 2019). Deficiency
of Fmt (slow growth phenotype) can be rescued by overproducing i-tRNA
(Nilsson et al. 2006; Shah et al. 2019; Shetty et al. 2016) suggesting
that in initiation, the role of formylation is primarily at the early
stages of i-tRNA binding to ribosome (Shetty and Varshney 2016).

In our genetic analyses, one of the suppressor strains (named B2) that
allowed initiation with 3GC mutant i-tRNA revealed a mutation
in\emph{fmt} gene (\emph{fmt} \textsubscript{am274}) wherein a Gln codon
(CAG) at position 274 was changed to an amber (UAG) codon, 42 codons
prior to the naturally occurring termination codon. Earlier biochemical
studies had shown that a deletion of even 20 amino acids from the
C-terminal, rendered Fmt inactive (Gite et al. 2000; Gite and
RajBhandary 1997; Ramesh, Gite, and RajBhandary 1998). Surprisingly,
analysis of the B2 suppressor showed that it produced full length Fmt,
albeit to a level of less than 5\% of the wild type strain. Given that
the B2 suppressor was derived from \emph{E. coli} KL16 lacking any
elongator tRNA derived amber suppressors, it revealed that the 3GC
mutant i-tRNA\textsubscript{CUA} (the only tRNA in cell with an amber
reading anticodon) read the amber codon at position 274 to produce full
length Fmt. We showed that diminished levels of cellular Fmt failed to
quantitatively formylate i-tRNA in real time, and the unformylated
i-tRNA (including the 3GC mutant) bound with EFTu to participate at the
step of elongation (in spite of it having a CxA mismatch at the top of
the acceptor stem). The B2 suppressor has evolved with an intriguing
mechanism of autoregulation to ensure balanced availability of both the
formylated and unformylated forms of i-tRNA (Shah et al. 2019). Any
excess production of Fmt would formylate increased levels of i-tRNA to
decrease the availability of unformylated i-tRNA to bind to EFTu (and to
suppress amber codon in the \emph{fmt} \textsubscript{am274} mRNA)
lowering Fmt production, which in turn would result in restoring
sufficient unformylated i-tRNA to bind to EFTu \textbf{(Fig. 5)} . The
lack of quantitative formylation in real time in B2 explains how i-tRNA
participates at the step of elongation. However, how does it allow
initiation with the 3GC mutant i-tRNA, especially because the deficiency
of formylation that applies to the cellular i-tRNA (wild type) will also
apply to the 3GC mutant i-tRNA? We believe that a critical level of
fMet-i-tRNA is required to saturate the available 30S in the cell.
Deficiency of the formylated form of the cellular i-tRNA leaves behind a
population of ribosomes which can now bind formylated fraction of the
3GC mutant i-tRNA (in the P-site) to allow its participation in
initiation (\textbf{Fig. 3, iii} ).

Formylation of i-tRNA depends on the availability of both the Fmt and
N\textsuperscript{10}-fTHF. As already shown decreased Fmt confers dual
functionality to i-tRNA and allows initiation with the 3GC mutant
i-tRNA. Would decreased levels of cellular N\textsuperscript{10}-fTHF
also confer a similar phenotype? N\textsuperscript{10}-fTHF is produced
via OCM by the action of a bifunctional enzyme, folate
dehydrogenase-cyclohydrolase (FolD), an essential enzyme in \emph{E.
coli} . FolD converts 5, 10 CH\textsubscript{2}-THF to 5, 10 CH-THF (by
its dehydrogenase activity), which is then converted to
N\textsuperscript{10}-fTHF (by its cyclohydrolase activity). In our
genetic screen, we isolated three different strains having point
mutations in their \emph{folD} genes viz., G122D (Das et al. 2008), C58Y
(Sah and Varshney 2015), and P140L (Lahry et al. 2020) compromising the
FolD activities, lowering the levels of N\textsuperscript{10}-fTHF and
the rate of formylation of i-tRNAs (Lahry et al. 2020). The FolD mutants
mimicked the phenotype of the strain with Fmt deficiency (B2
suppressor). Interestingly, the strength of the phenotype correlated
directly with the deficiency of FolD activity of the alleles (Lahry et
al. 2020). Also, inhibitors of FolA (dihydrofolate reductase, which
converts DHF to THF) such as trimethoprim (TMP) led to decreased levels
of formylation of i-tRNA and in loss of fidelity of initiation allowing
initiation with the 3GC mutant i-tRNA. Interestingly, we noticed that
mutant \emph{folD} strains were hypersensitive to TMP. Likewise, our
earlier investigations with the \emph{folD122} allele (FolD, G122D)
showed that the strain suffered from the deficiencies of Met and
S-adenosylmethionine (SAM). A deficiency of SAM would result in
deficiency of methylations in rRNA. We showed that deletion of
methyltransferases that methylate 16S rRNA do lead to compromised
fidelity of i-tRNA selection on the ribosome (Das et al. 2008).

The mechanism underlying the dual function of mammalian mitochondrial
tRNA\textsuperscript{Met} at both the initiation and elongation steps
was thought to be by incomplete formylation of this tRNA population
(Takeuchi et al. 1998, 2001). Thus, the fact that i-tRNA functions both
at the initiation and elongation steps in the Fmt or FolD deficient
strains (Lahry et al. 2020; Shah et al. 2019) inspired us to use i-tRNA
mutants deficient in formylation to sustain \emph{E. coli} for its total
load of initiation (all 4 i-tRNA genes, \emph{metZWV} and
\emph{metY}were deleted) and elongation (both elongator
tRNA\textsuperscript{Met}genes, \emph{metT} and \emph{metU} were
deleted). This study led to identification of i-tRNA mutants that
resembled human mitochondrial i-tRNA in their acceptor stem sequences
(Govindan et al. 2018).

\textbf{(iii) Role of ribosomal components, ribosome biogenesis factors,
translation factors and their coevolution in the fidelity of initiation
and/or ribosome biogenesis.}

(a) 16S rRNA (anti Shine-Dalgarno sequence) and the accuracy of i-tRNA
selection in P-site: The interaction between the Shine-Dalgarno (SD)
sequence often found upstream of the initiation codon in mRNAs, and
anti-SD (aSD) sequence in the 3' end of 16S rRNA, facilitates binding
and placement of mRNA on 30S, and this interaction has a significant
impact on the efficiency of initiation. An optimal strength of SD-aSD
interaction is crucial for efficient and accurate initiation. Though
previously believed that a high affinity between SD-aSD promotes
translation initiation (Lim, Furuta, and Kobayashi 2012; Ma, Campbell,
and Karlin 2002; Schurr, Nadir, and Margalit 1993; Starmer et al. 2006),
it was later suggested that a sub-optimal level of SD-aSD affinity,
which often occurs in \emph{E. coli} , increases the efficiency of
translation (Osterman et al. 2013; Wei, Silke, and Xia 2017). The
correlation between the length of the SD sequence and the efficiency of
translation follows a bell-shaped curve, with a maximum initiation
efficiency in the range of 4-6 base pairs (Chen et al. 1994; Ma et al.
2002; Vimberg et al. 2007). Our suppressor analysis revealed that a C to
T mutation at position 1536 towards the 3' end of the 16S rRNA
(in\emph{rrsC} operon) resulting in an extended SD-aSD interaction of 8
bp (as opposed to the original 6 bp) with the reporter
CAT\textsubscript{am1} mRNA, impaired scrutiny of i-tRNA, enabling
initiation with the 3GC mutant i-tRNA (Shetty et al. 2014). This
extended SD-aSD interaction of 8 bp mimics the conditions used in
determining the X-ray crystal structure of the bacterial ribosome bound
to the fMet-i-tRNA and mRNA (Selmer et al. 2006) wherein a weaker
interaction of G1338 and A1339 was observed with the 3GC pairs. The
extended SD-aSD interaction triggers a \textgreater{}2 movement between
the h26 and h28 of 16S rRNA, resulting in a change~in the position of
G1338 and A1339 (Korostelev et al. 2007; Selmer et al. 2006; Shetty et
al. 2014). Thus, the SD-aSD context and its extent of the base pairing
decide not only the rate of initiation but also the fidelity of
initiation.

(b) Importance of ribosome maturation in accuracy of initiation: In the
section on `Initiator tRNA abundance and ribosome maturation', we
described a novel role of i-tRNA particularly the 3GC pairs during the
pioneering round of initiation, in the terminal stages of ribosome
maturation. The work showed that the ribosomes when not completely
mature (for example because of deletion of \emph{metZWV} ) use the 3GC
mutant i-tRNA for initiation. Ribosome biogenesis/maturation is an
elaborate process requiring RNA/RNA, RNA/protein, and protein/protein
interactions to process precursor rRNA molecules, nucleoside
modifications, structural rearrangements, and interaction of r-proteins.
A number of biogenesis factors are also required to assemble mature
ribosomes \emph{in vivo} (Kaczanowska and Ryd\selectlanguage{ngerman}én-Aulin 2007; Shajani,
Sykes, and Williamson 2011). Interestingly, one of the suppressor
strains (named A18) allowed us to uncover a new role for RluD in
ribosome biogenesis and fidelity of initiation. RluD binds 50S and
modifies U1911, U1915, and U1917 in H69 of 23S rRNA to pseudouridines.~
In A18, RluD suffered an E265K mutation in its C-terminal tail. This
mutation causes no detectable change in the biochemical activity of RluD
as pseudouridine synthase but allows initiation with the 3GC mutant
i-tRNA (Lahry et al. 2022). An earlier study where a catalytically dead
RluD was used, inferred an alternate function of RluD in rescue
of\emph{E. coli} growth (Gutgsell et al. 2001). The RluD (E265K) results
in increased association of RbfA to 30S. The data suggested that the
C-terminal tail of RluD facilitates release of RbfA from the
30S\textbf{(Fig. 4)} . The findings not only suggest the role of RluD in
the fidelity of translation initiation via promoting 30S biogenesis but
also resolve a long-held hypothesis of the alternate function of RluD.
RbfA bound to 30S may affect the scrutiny of the 3GC pairs in the P-site
possibly via G1338 and A1339 (Korostelev et al. 2007; Shetty et al.
2014) to allow initiation even with an i-tRNA mutated in its 3GC
sequence. Interestingly, IF3 has also been shown to release RbfA from
30S (Sharma and Woodson 2020).

(c) Initiation factors in accuracy of i-tRNA selection: Initiation is
assisted by three essential initiation factors, IF1, IF2, and IF3 which
scrutinize the fidelity of translation initiation. The ribosome-bound
IF2 recruits i-tRNA to 30S (Milon et al. 2010). IF1 prevents untimely
access of tRNAs to the A-site and aids in positioning of IF2 bound
i-tRNA in the P-site in P/I (the peptidyl/initiation) state, forming the
30S PIC (Antoun et al. 2006b, 2006a). Although IF1 and IF2 are
independent proteins in bacteria, we showed that an insertion of 37
amino acids in IF2 (in mammalian mitochondria) substitutes for the
independent requirement of IF1 in \emph{E. coli}(Gaur et al. 2008). In
case of IF3, it has been shown that it not only serves as an
anti-association factor (preventing association of 30S and 50S in the
absence of an mRNA) but also as a proof-reader of the 3GC pairs in
i-tRNA on the 30S P-site (Ayyub, Dobriyal, and Varshney 2017; O'Connor
et al. 2001). Biochemical analysis suggested that all of its (IF3) known
activities (anti-association of the ribosomal subunits, prevention of
pseudo-initiation complex formation, and prevention of initiation from
leaderless mRNAs and non-canonical initiation codons) can be discharged
by the C-terminal globular domain (CTD) alone (Dallas and Noller 2001;
Godefroy-Colburn et al. 1975; Gualerzi and Pon 1990; Hartz et al. 1990;
O'Connor et al. 2001; Singh et al. 2005; Tedin et al. 1999), and the
globular N-terminal domain (NTD) was deemed to provide stability of
binding to IF3 (Petrelli et al. 2001). Our \emph{in vivo}studies on the
individual domains led to characterization of a crucial role of the NTD
in the fidelity i-tRNA selection (Ayyub et al. 2017). Subsequently, we
showed that IF3 NTD interactions with the elbow region of i-tRNA are
crucial for the movements of the two domains of IF3 and in the fidelity
of P-site binding of i-tRNA (Singh et al. 2022). Other studies revealed
that the genetic interactions between uS12 and IF3 also play a role in
i-tRNA selection (Datta, Singh, et al. 2021).

(d) Coevolution of the translation apparatus: Initiator tRNAs in
mycoplasma and rhizobia species possess variations in the 3GC sequence
in their i-tRNA anticodon stems. For example, variations like
A\textsubscript{29}-U\textsubscript{41},
G\textsubscript{30}-C\textsubscript{40},
G\textsubscript{31}-C\textsubscript{39} (AU/GC/GC);
G\textsubscript{29}-C\textsubscript{41},
G\textsubscript{30}-C\textsubscript{40},
G\textsubscript{31}-U\textsubscript{39} (GC/GC/GU) or
A\textsubscript{29}-U\textsubscript{41},
G\textsubscript{30}-C\textsubscript{40},
G\textsubscript{31}-U\textsubscript{39} (AU/GC/GU), are of common
occurrence in these organisms \textbf{(Fig. 6A)} . Computational
analysis showed that unconventional nature of the 3GC pairs in these
i-tRNAs is accompanied with changes in other components of the
translation apparatus. The conserved C-terminal tail (SKR) of uS9 is
represented by TKR sequence in mycoplasma, and uS13 possesses a longer
C-terminal tail. A conserved R131 position in IF3, is represented by P,
F or Y in mycoplasma harbouring i-tRNA with AU/GC/GU pairs. Initiation
with non-AUG codons is also common in mycoplasma. When these features
(P131 in IF3, TKR at C-terminal tail of uS9, and longer C-terminal tail
of uS13) were tested in \emph{E. coli} (Ayyub et al. 2018), they
facilitated initiation with i-tRNA\textsubscript{AU/GC/GU} and 3GC
mutant i-tRNA.

Additional understanding of coevolution of the translation apparatus,
particularly the interplay between i-tRNA, IF3 and uS12 came from the
homologous system of \emph{E. coli} . We earlier showed that \emph{E.
coli} lacking native i-tRNA genes could be sustained on
i-tRNA\textsubscript{cg/GC/cg} \textbf{(Fig. 6A)} . However, these
strains grew poorly (Shetty et al. 2016). We later discovered that for
sustenance on i-tRNA\textsubscript{cg/GC/cg} as the only source of
i-tRNA, \emph{E. coli} required V93A mutation in IF3. When this slow
growing \emph{E. coli} strain was repeatedly sub-cultured to acquire
suppressor mutations to enable faster growth, it came up with additional
mutations of either V32L or H76L in uS12. The V93A mutation in IF3 was
the initial requirement to decrease the stringency of i-tRNA selection
at the P-site, essential for survival with the only available
i-tRNA\textsubscript{cg/GC/cg}, at the cost of compromised overall
growth rate. The succeeding mutations in the genome that enhance growth,
occurred in a r-protein, uS12 (V32L or H76L). This led to the discovery
of cooperation between uS12, IF3 and i-tRNA for faithful initiation and
a crosstalk between uS12, RRF, EFG and Pth (recycles tRNAs from
peptidyl-tRNAs by hydrolysing the ester bond between the peptide and
tRNA) modulating ribosome recycling to fine-tune the overall fidelity of
translation initiation (Datta, Pillai, et al. 2021). The aforementioned
systematic evolution demonstrated a paradigm for coevolution of the
translation apparatus (\textbf{Fig. 6B} ).

(e) Ribosome recycling factor and the fidelity of initiation: Ribosome
recycling, a vital step in protein synthesis, dissociates the
post-termination complexes to make the ribosomal subunits available for
another round of initiation (Kiel, Kaji, and Kaji 2007). We and others
showed that the specific interactions between RRF and EFG, the two key
factors involved at this step, are crucial in dissociation of the
complexes consisting of mRNA-bound ribosomes harbouring deacylated tRNA
(Fujiwara et al. 2004; Ito et al. 2002; Rao and Varshney 2001).
Subsequently, it was shown that IF3 also plays an active role in
ribosome recycling (Singh et al. 2005). The genetic interactions between
RRF and Pth (Das and Varshney 2006) were seen in several independent
investigations (Seshadri et al. 2009; Singh et al. 2005, 2008; Singh and
Varshney 2004). These interactions strongly suggest that RRF, EFG and
IF3 can act together on the pre-termination ribosomal complexes to
release peptidyl-tRNAs. On the other hand, the structural investigations
inferred that pre-termination ribosomal complexes are not optimal
substrates for RRF binding (Schmeing and Ramakrishnan 2009; Weixlbaumer
et al. 2008). However, given that RRF levels are high in cell
(\textasciitilde{}20 \selectlanguage{greek}µ\selectlanguage{english}M) (Prabhakar et al. 2017), and multiple GTP
molecules are often hydrolysed in carrying out a single round of
recycling (Borg et al. 2016), it is feasible that the pre-termination
ribosomal complexes, at least the ones carrying short peptidyl-tRNAs (e.
g., at the interface of initiation and elongation steps or in early
elongation), may be utilized as substrates by RRF (\textbf{Fig. 7} ).

In this context, would recycling of the incorrectly assembled 70S
complexes (carrying formyl-aminoacyl-i-tRNA) or the ones wherein the
initial codons in the ORF were incorrectly decoded, by RRF increase the
accuracy of initiation? Evidence for this came from our earlier studies
where we genetically combined the \emph{folD122} allele (having G122D
mutation in FolD) with the \emph{frr} \textsuperscript{ts} allele
(temperature sensitive allele of RRF from \emph{E. coli} LJ14) (Seshadri
et al. 2009). As already described, \emph{folD122} allows initiation
with the 3GC mutant i-tRNA. Interestingly, when we transduced \emph{E.
coli} LJ14 strain (reduced level of RRF) with \emph{folD122} allele, we
noted increased initiation with the 3GC mutant i-tRNA, suggesting that
deficiency of RRF allowed increased accommodation of the 3GC mutant
i-tRNA in the ribosomal P-site (Singh et al. 2008) showing that RRF
plays a role in accuracy of i-tRNA selection. Furthermore, we noted that
uS12 (through its PNSA loop), shows genetic interaction with RRF and
Pth, which are important in the fidelity of translation (Datta, Pillai,
et al. 2021).

More importantly, as mentioned, in our coevolution study involving
initiation with the i-tRNA\textsubscript{cg/GC/cg}, we noted that in the
background of IF3 (V93A), while H76L mutation in uS12 improved the
accuracy of i-tRNA selection, the V32L mutation compensated for the
reduced fidelity of i-tRNA selection by ensuring a fidelity check by
enhanced RRF function (Datta, Singh, et al. 2021). This investigation
highlighted the importance of the genetic interactions between i-tRNA,
IF3, uS12, EF-G, RRF, and Pth in maintaining the overall fidelity of
initiation. It is satisfying that a more recent investigation further
supports the role of RRF in peptidyl-tRNA drop-off in early stages of
elongation as an additional quality control measure to maintain faithful
translation (Nagao et al. 2023).

\textbf{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
N\textsuperscript{10}-fTHF) of the cell would favour translation
initiation, and an energy depleted state (lower flux of Met and
N\textsuperscript{10}-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
tRNA\textsuperscript{Met} (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
A\textsubscript{1}:U\textsubscript{72}, and the structural features in
T\selectlanguage{greek}ψ\selectlanguage{english}C arm of mammalian i-tRNA, and the 2'-O-phosphoribosyl modification at
position 64 in yeast i-tRNA avoid i-tRNA binding to eEF1A (Desgr\selectlanguage{ngerman}ès 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) \textbf{(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.

\textbf{Conflicts of interest}

The authors declare that they have no conflicts of interest with the
contents of this article.

\textbf{Acknowledgments}

The authors thank Dr. Sunil Shetty for his feedback on the manuscript.

\textbf{Funding}

Research in the area of protein synthesis in UV's laboratory was
supported by grants from Science \& Engineering Research Board,
(EMR/2016/005617); Department of Biotechnology,
(BT/PR28058/BRB/10/1674/2018, BT/PR13522/COE/34/27/2015), New Delhi;
Jamsetji Tata Trust {[}R(HR)Tata Chairs/2020-854/78{]}, and the J. C.
Bose Fellowship.

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\textbf{Figures legends:}

\textbf{Fig. 1: An overview of the different steps in bacterial
translation.} The i-tRNA and mRNA assemble on 30S ribosome bound with
IF1, IF2 and IF3 to form 30S pre-initiation complex (PIC) which then
converts into 30S IC. A circle on each tRNA indicates charging by the
cognate amino acid and the star indicates its formylation. The i-tRNA
transits from a P/I to a P/P state during its accommodation in the 70S
resulting in an elongation competent 70S complex. The 70S complex then
enters the repetitive cycles of peptide bond formation to extend the
peptide chain by one amino acid each time with the help of elongation
factors. When the A-site is presented with a stop codon in the mRNA,
termination occurs with the help of release factors 1/ 2 and 3,
releasing the nascent protein. The mRNA bound ribosome harbouring
deacylated tRNA is then recycled by the concerted action of EF-G, RRF
and IF3.

\textbf{Figure 2: (A) Clover-leaf structures of i-tRNA, 3GC mutant
i-tRNA, and elongator tRNA\textsuperscript{Met}.} (i) fMet-i-tRNA, (ii)
fGln-i-tRNA\textsubscript{CUA/ua/cg/au}, (iii)
Met-tRNA\textsuperscript{Met}. The unique structural features of i-tRNA
are highlighted. For details, see the text. \textbf{(B)}\emph{In vivo}
\textbf{assay for initiation and isolation of suppressor strains that
allow initiation with the 3GC mutant i-tRNA.} (i) \emph{E. coli}
harbouring pCAT\textsubscript{am1} produces
CAT\textsubscript{am1}reporter mRNA but it cannot be translated as the
cells lack i-tRNA to pair with the UAG start codon and the cells are
Cm\textsuperscript{S}. (ii) \emph{E. coli} harbouring
pCAT\textsubscript{am1}\emph{metY} \textsubscript{CUA} make
CAT\textsubscript{am1} mRNA, and i-tRNA\textsubscript{CUA} (from the
CAT\textsubscript{am1} and \emph{metY} \textsubscript{CUA} gene present
on the plasmid). The CUA anticodon of i-tRNA\textsubscript{CUA} pairs
with UAG initiation codon in CAT\textsubscript{am1} mRNA, and
translation of CAT\textsubscript{am1} mRNA results in production of CAT
to confer Cm\textsuperscript{R} to the cells. (iii) \emph{E.
coli}harbouring pCAT\textsubscript{am1}\emph{metY}
\textsubscript{CUA/ua/cg/au} makes CAT\textsubscript{am1} reporter mRNA
and the 3GC mutant i-tRNA (i-tRNA\textsubscript{CUA/ua/cg/au}) from the
respective genes but because of the mutations in the 3GC pairs, the
i-tRNA\textsubscript{CUA/ua/cg/au} fails to initiate from the UAG
initiation codon of CAT\textsubscript{am1} mRNA and the cells are
Cm\textsuperscript{S}. (iv) Same as (iii) except that a suppressor
mutation in the host genome (yellow asterisk) facilitates
i-tRNA\textsubscript{CUA/ua/cg/au} to initiate from the UAG start codon
of CAT\textsubscript{am1} mRNA and the cells become
Cm\textsuperscript{R}.

\textbf{Figure 3: Fidelity of initiation depends on the availability of
a `critical level' of fMet-i-tRNA} . \textbf{(i)} In wild type \emph{E.
coli} an adequate amount of Met-i-tRNA is available, which is rapidly
formylated to fMet-i-tRNA by the sufficiency of
N\textsuperscript{10}-fTHF and Fmt. The presence of fMet-i-tRNA above a
critical threshold ensures occupancy of P-sites on all the available 30S
avoiding binding of non-canonical i-tRNAs disallowing initiation with
them. \textbf{(ii)} A strain in which expression of canonical i-tRNAs is
reduced, or \textbf{(iii)} Met-i-tRNA is not formylated in real time due
to the decreased levels of Fmt or N\textsuperscript{10}-fTHF, the amount
of available fMet-i-tRNA is inadequate (below critical level) to occupy
all available 30S P-sites, failing to avoid binding of non-canonical
i-tRNAs (or elongator tRNAs) and initiation with them.

\textbf{Figure 4: Ribosome biogenesis in the fidelity of translation
initiation:} In wild type \emph{E. coli} , a canonical i-tRNA (with
intact 3GC pairs) facilitates the ultimate steps of maturation of 16S
rRNA (in 30S) in 70S ribosome by inducing trimming of the extra
sequences at the 5' and the 3' ends by appropriate RNases. Conversely,
the binding of non-canonical 3GC mutant i-tRNA with wild type anticodon,
CAU blocks the final maturation of 16S rRNA leading to immature
ribosomes. Similarly, RluD plays a crucial role in efficient release of
RbfA from 30S subunit. However, the mutant
RluD\textsubscript{E265K}fails in efficient release of ribosome binding
factor A (RbfA) from 30S and allows translation initiation with
non-canonical i-tRNAs (3GC mutant i-tRNA).

\textbf{Figure 5: Role of Fmt in participation of i-tRNA at the steps of
initiation and elongation.} In wild type bacteria, expression of Fmt
facilitates rapid formylation of i-tRNAs and their preferential
participation in initiation. However, the reduced level of Fmt leads to
slow formylation of i-tRNAs resulting in availability of unformylated
i-tRNA population. The unformylated i-tRNA can bind with EF-Tu and
participate at the step of elongation. Therefore, enough Fmt levels are
important to avoid involvement of i-tRNA in elongation.

\textbf{Fig. 6:} \textbf{Coevolution of the translation apparatus to
optimize translation initiation with i-tRNA variants. A.} Clover leaf
structure of the i-tRNA in \emph{E. coli} highlighting the 3GC pair
(light olive green). \emph{Mycoplasma} sp. harbour three variants of the
3GC pairs in the anticodon stem namely
A\textsubscript{29}-U\textsubscript{41},
G\textsubscript{30}-C\textsubscript{40},
G\textsubscript{31}-C\textsubscript{39} (AU/GC/GC);
G\textsubscript{29}-C\textsubscript{41},
G\textsubscript{30}-C\textsubscript{40},
G\textsubscript{31}-U\textsubscript{39} (GC/GC/GU) or
A\textsubscript{29}-U\textsubscript{41},
G\textsubscript{30}-C\textsubscript{40},
G\textsubscript{31}-U\textsubscript{39} (AU/GC/GU). Another i-tRNA
mutant investigated contained c\textsubscript{29}-g\textsubscript{41},
G\textsubscript{30}-C\textsubscript{40},
c\textsubscript{31}-g\textsubscript{39} (cg/GC/cg) in the anticodon
stem. \textbf{B.} Cryo-EM structure (PDB ID:5LMQ) of the mRNA (blue)
bound 30S PIC (grey) along with IF3 (cyan) highlighting the relative
molecular positions of the ribosomal proteins shown to directly (uS13 in
red and uS9 in green) or indirectly (uS12 in magenta) play a role in
moderating the fidelity of initiation by scrutinizing the 3GC pairs of
the i-tRNA (brick-red) bound at the ribosomal P-site. The 16S rRNA is
depicted in grey. Structure modified using PyMOL software.

\textbf{Fig. 7: Fidelity of initiation by recycling of ribosomes at the
elongation competent 70S complex stage or early in the elongation
step.}The canonical pathway of initiation and its transition into the
elongation step (grey arrows) follows the stages of 30S IC formation and
its transition into the 70S complex and the early stages of elongation
cycles. However, in the cases where translation proceeded with the
incorrectly assembled 70S complex or errors arising in early stages of
elongation, the ribosomes may become a substrate for disassembly by the
action of RRF, EFG and IF3 (orange dashed arrows).

\textbf{Fig. 8: The initiator tRNA-centric view of faithful
translation.} Schematics showing that under the conditions of
sufficiency of i-tRNA, Fmt and N\textsuperscript{10}-fTHF levels, the
translation initiation occurs with high fidelity. However, under the
deficiency of one or more of these (i-tRNA, Fmt or
N\textsuperscript{10}-fTHF) the fidelity of initiation is relaxed to
allow initiation with the non-canonical i-tRNAs or elongator tRNAs. The
consequences of the high and compromised fidelity of initiation have
been indicated.

\textbf{Hosted file}

\verb`Suppressor review_figures_Final_19-12-23.pptx`  available at \url{https://authorea.com/users/759312/articles/733821-fidelity-of-translation-initiation-in-bacteria-an-initiator-trna-centric-view}

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