Figures legends:
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.
Figure 2: (A) Clover-leaf structures of i-tRNA, 3GC mutant
i-tRNA, and elongator tRNAMet. (i) fMet-i-tRNA, (ii)
fGln-i-tRNACUA/ua/cg/au, (iii)
Met-tRNAMet. The unique structural features of i-tRNA
are highlighted. For details, see the text. (B)In vivo assay for initiation and isolation of suppressor
strains that allow initiation with the 3GC mutant i-tRNA. (i) E.
coli harbouring pCATam1 produces CATam1reporter mRNA but it cannot be translated as the cells lack i-tRNA to
pair with the UAG start codon and the cells are CmS.
(ii) E. coli harbouring
pCATam1metY CUA make
CATam1 mRNA, and i-tRNACUA (from the
CATam1 and metY CUA gene present
on the plasmid). The CUA anticodon of i-tRNACUA pairs
with UAG initiation codon in CATam1 mRNA, and
translation of CATam1 mRNA results in production of CAT
to confer CmR to the cells. (iii) E. coliharbouring
pCATam1metY CUA/ua/cg/au makes
CATam1 reporter mRNA and the 3GC mutant i-tRNA
(i-tRNACUA/ua/cg/au) from the respective genes but
because of the mutations in the 3GC pairs, the
i-tRNACUA/ua/cg/au fails to initiate from the UAG
initiation codon of CATam1 mRNA and the cells are
CmS. (iv) Same as (iii) except that a suppressor
mutation in the host genome (yellow asterisk) facilitates
i-tRNACUA/ua/cg/au to initiate from the UAG start codon
of CATam1 mRNA and the cells become
CmR.
Figure 3: Fidelity of initiation depends on the availability of
a ‘critical level’ of fMet-i-tRNA . (i) In wild type E.
coli an adequate amount of Met-i-tRNA is available, which is rapidly
formylated to fMet-i-tRNA by the sufficiency of
N10-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. (ii) A strain in which expression of canonical i-tRNAs is
reduced, or (iii) Met-i-tRNA is not formylated in real time due
to the decreased levels of Fmt or N10-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.
Figure 4: Ribosome biogenesis in the fidelity of translation
initiation: In wild type 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 RluDE265Kfails in efficient release of ribosome binding factor A (RbfA) from 30S
and allows translation initiation with non-canonical i-tRNAs (3GC mutant
i-tRNA).
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.
Fig. 6: Coevolution of the translation apparatus to
optimize translation initiation with i-tRNA variants. A. Clover leaf
structure of the i-tRNA in E. coli highlighting the 3GC pair
(light olive green). Mycoplasma sp. harbour three variants of the
3GC pairs in the anticodon stem namely
A29-U41,
G30-C40,
G31-C39 (AU/GC/GC);
G29-C41,
G30-C40,
G31-U39 (GC/GC/GU) or
A29-U41,
G30-C40,
G31-U39 (AU/GC/GU). Another i-tRNA
mutant investigated contained c29-g41,
G30-C40,
c31-g39 (cg/GC/cg) in the anticodon
stem. 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.
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).
Fig. 8: The initiator tRNA-centric view of faithful
translation. Schematics showing that under the conditions of
sufficiency of i-tRNA, Fmt and N10-fTHF levels, the
translation initiation occurs with high fidelity. However, under the
deficiency of one or more of these (i-tRNA, Fmt or
N10-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.