Rapid formylation of the cellular initiator tRNA population makes a crucial contribution to its exclusive participation at the step of initiation

Abstract

Initiator tRNAs (i-tRNAs) possess highly conserved three consecutive GC base pairs (GC/GC/GC, 3GC pairs) in their anticodon stems. Additionally, in bacteria and eukaryotic organelles, the amino acid attached to i-tRNA is formylated by Fmt to facilitate its targeting to 30S ribosomes. Mutations in GC/GC/GC to UA/CG/AU in i-tRNA CUA/ 3GC do not affect its formylation. However, the i-tRNA CUA/ 3GC is non-functional in initiation. Here, we characterised an Escherichia coli strain possessing an amber mutation in its fmt gene ( fmt am274 ), which affords initiation with i-tRNA CUA/ 3GC . Replacement of fmt with fmt am274 in the parent strain results in production of truncated Fmt, accumulation of unformylated i-tRNA, and a slow growth phenotype. Introduction of i-tRNA CUA/ 3GC into the fmt am274 strain restores accumulation of formylated i-tRNAs and rescues the growth defect of the strain. We show that i-tRNA CUA/ 3GC causes a low level suppression of am274 in fmt am274 . Low levels of cellular Fmt lead to compromised efficiency of formylation of i-tRNAs, which in turn results in distribution of the charged i-tRNAs between IF2 and EF-Tu allowing the plasmid borne i-tRNA CUA/ 3GC to function at both the initiation and elongation steps. We show that a speedy formylation of i-tRNA population is crucial for its preferential binding (and preventing other tRNAs) into the P-site.

The Variety in the Common Theme of Translation Inhibition by Type II Toxin–Antitoxin Systems

“…Subsets of toxins sharing similar target sequences are boxed with different colors. Protein identifiers and cleavage specificity were taken from: Haemophilus influenzae VapC1 Hin (NP_438487.1), VapC2 Hin (NP_439108.1) (Walling and Butler, 2018); Salmonella enterica VapC LT2 (NP_461950.1), Shigella flexneri MvpT (AMN61047.1) (Winther and Gerdes, 2011); Leptospira interrogans VapC Lin (AAS71220.1) (Lopes et al, 2014); Mycobacterium tuberculosis VapC-mt4 (NP_215109.1) (Cruz et al, 2015;Winther et al, 2016), VapC-mt11 (NP_216077.1) (Winther et al, 2016;Cintron et al, 2019), VapC-mt15(NP_216526.1), VapC-mt25 (NP_214791.1), VapC-Mt28 (NP_215123.1), VapC-mt29 (NP_215131.1), VapC-mt30(NP_215138.1), VapC-mt32(NP_215630.1), VapC-mt33 (NP_215758.1), VapC-mt37 (NP_216619.1), VapC-mt39 (NP_217046.1) (Winther et al, 2016), VapC-mt20 (NP_217065.1), VapC-mt26 (NP_215096.1) (Winther et al, 2013(Winther et al, , 2016 growth (Shah et al, 2019) and is impaired by the Met acetylation, resulting in a strong growth inhibition and generating a dead-end product acMet-tRNA fMet . In vivo expression of AtaT manifests in accumulation of ribosome assembly intermediates, reflecting a strong inhibition of translation initiation (Jurenas et al, 2017a).…”

Regulation of translation by one-carbon metabolism in bacteria and eukaryotic organelles

“…Formylation of Met-tRNA fMet may be more crucial for initiation with 70S ribosomes as opposed to the canonical form of initiation that begins with 30S subunits ( 101 , 102 ). The deletion of the Fmt gene in E. coli drastically reduces its growth ( 100 , 103 , 104 , 105 ). However, formylation was found to be dispensable in other bacteria such as Pseudomonas, Streptococci, and Bacillus species ( 106 , 107 ).…”

“…To understand the importance of formylation, we have used a mutant tRNA fMet called the “3GC mutant” wherein the highly conserved 3GC base pairs in the anticodon stem were substituted by the sequence found in the elongator tRNA Met as a model to investigate the fidelity of tRNA selection at the step of initiation ( 143 ). Using this model, we observed that an E. coli mutant expressing low levels of Fmt (consequently a low rate of formylation) permitted initiation with the 3GC mutant tRNA ( 105 ). As Fmt expression was increased, participation of the 3GC mutant tRNA in initiation was diminished.…”

Hijacking Translation Initiation for Synthetic Biology

“…As the latter is chemically equivalent to a peptidyl‐tRNA, the mismatch (C1xA72) is also essential for protecting the aminoacyl‐tRNA from hydrolysis by peptidyl‐tRNA hydrolase . Formylation of Met‐tRNA fMet has two functions: 1) precluding fMet‐tRNA fMet from elongation, and 2) increasing the affinity of the aminoacyl‐ i tRNA for initiation factor 2 (IF2) …”

“…Because of its early origin, the amino acid used for initiation must have been able to be synthesized via one‐carbon metabolism. Met and N 10 ‐formyltetrahydrofolate, the precursors of fMet, are perfect candidates providing a possible explanation for Met as the initiating amino acid …”