(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, N10-fTHF (Fig. 3,
i ). Interestingly, Fmt can also utilize N10-formyl
dihydrofolate (N10-fDHF) as a poor alternate for
N10-fTHF (Sah and Varshney 2023). The deletion offmt (gene encoding Fmt) causes modest to severe growth defects inPseudomonas aeruginosa and 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 infmt gene (fmt 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 E. coli KL16 lacking any
elongator tRNA derived amber suppressors, it revealed that the 3GC
mutant i-tRNACUA (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 fmt am274 mRNA)
lowering Fmt production, which in turn would result in restoring
sufficient unformylated i-tRNA to bind to EFTu (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 (Fig. 3, iii ).
Formylation of i-tRNA depends on the availability of both the Fmt and
N10-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 N10-fTHF
also confer a similar phenotype? N10-fTHF is produced
via OCM by the action of a bifunctional enzyme, folate
dehydrogenase-cyclohydrolase (FolD), an essential enzyme in E.
coli . FolD converts 5, 10 CH2-THF to 5, 10 CH-THF (by
its dehydrogenase activity), which is then converted to
N10-fTHF (by its cyclohydrolase activity). In our
genetic screen, we isolated three different strains having point
mutations in their 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 N10-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 folD strains were hypersensitive to TMP. Likewise, our
earlier investigations with the 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
tRNAMet 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 E. coli for its total
load of initiation (all 4 i-tRNA genes, metZWV and metYwere deleted) and elongation (both elongator tRNAMetgenes, metT and 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).