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.