The i6A37 tRNA modification is essential for proper decoding of UUX-Leucine codons during rpoS and iraP translation

Aubee, Joseph I; Olu, Morenike; Thompson, Keyata N.

doi:10.1261/rna.053165.115

Cited by 31 publications

(52 citation statements)

References 63 publications

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“…Such effects of specific tRNA modifications on codon translation could account for the different codon pair biases observed in species that are evolutionarily distant (possessing different specific tRNA modifications) and also could account for the difficulty in expressing proteins in heterologous systems, i.e., expressing proteins from plant and mammalian systems in bacteria. The MiaA (i6A37) modification has recently been shown to affect mRNA translation in E. coli in a codon context-dependent manner, supporting our overall hypothesis (20). The translation of proline codons in the mgtL peptide transcript of Salmonella was recently shown to be affected by mutations defective in ribosomal proteins L27, L31, elongation factor EF-P, and TrmD, which catalyzes the m 1 G37 methylation of proline tRNA (21).…”

Section: Discussionsupporting

confidence: 80%

Case for the genetic code as a triplet of triplets

Chevance

Hughes

2017

Proc. Natl. Acad. Sci. U.S.A.

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The efficiency of codon translation in vivo is controlled by many factors, including codon context. At a site early in the Salmonella flgM gene, the effects on translation of replacing codons Thr6 and Pro8 of flgM with synonymous alternates produced a 600-fold range in FlgM activity. Synonymous changes at Thr6 and Leu9 resulted in a twofold range in FlgM activity. The level of FlgM activity produced by any codon arrangement was directly proportional to the degree of in vivo ribosome stalling at synonymous codons. Synonymous codon suppressors that corrected the effect of a translation-defective synonymous flgM allele were restricted to two codons flanking the translation-defective codon. The various codon arrangements had no apparent effects on flgM mRNA stability or predicted mRNA secondary structures. Our data suggest that efficient mRNA translation is determined by a triplet-of-triplet genetic code. That is, the efficiency of translating a particular codon is influenced by the nature of the immediately adjacent flanking codons. A model explains these codon-context effects by suggesting that codon recognition by elongation factor-bound aminoacyl-tRNA is initiated by hydrogen bond interactions between the first two nucleotides of the codon and anticodon and then is stabilized by base-stacking energy over three successive codons.translation | genetic code | context effects on translation | tRNA | synonymous codon effects T he DNA sequence is transcribed to form mRNA, which then is translated into protein by ribosomes. The genetic code consists of 64 triplet RNA codons that specify the 20 amino acids and sites of translation termination (stop codons). The decoding process involves interaction between a specific codon in the mRNA and the three-base anticodon of the cognate aminoacyltRNA (bases 36, 35, and 34 within the anticodon loop) (1). The code is redundant, in that many amino acids are specified by two or more triplet sequences. In addition, codons specifying a particular amino acid are recognized by single or multiple tRNAs. Most tRNA species recognize codons that differ in the third or "wobble" position. Modifications of tRNA bases also contribute to tRNA recognition of single or multiple anticodons as the tRNA enters the ribosome at the A site (1). Codons are read in the 5′-3′ direction of the mRNA by sequential interactions with tRNA aligned 3′-5′ vis-à-vis each codon. Stacking-related wobble codon recognition is mediated by extensive modification of position 34 of the tRNA codon.Other base modifications stabilize the interaction of the tRNA with ribosomal A site, i.e., methylation of a G at position 37 prevents tRNA slippage, which would result in a shift in the translation reading frame (1). Fig. S1 illustrates the initial translation steps starting with codon recognition and proceeding to peptidyl transfer. GTP-bound elongation factor Tu (EF-Tu) brings aminoacyl-tRNA to the A site of the ribosome. WatsonCrick pairing at the first two bases of the codon initiates EF-TutRNA docking followed by triplet sta...

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Section: Discussionsupporting

confidence: 80%

Case for the genetic code as a triplet of triplets

Chevance

Hughes

2017

Proc. Natl. Acad. Sci. U.S.A.

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“…In addition, there have only been a few predicted HULC proteins tested for MiaA sensitivity [19]. Demonstrating the i 6 A37 sensitivity for an additional HULC protein, hfq , strengthens the predictive power of this model [19].…”

Section: Discussionmentioning

confidence: 99%

TrmL and TusA Are Necessary for rpoS and MiaA Is Required for hfq Expression in Escherichia coli

2017

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Previous work demonstrated that efficient RNA Polymerase sigma S-subunit (RpoS) translation requires the N6-isopentenyladenosine i6A37 transfer RNA (tRNA) modification for UUX-Leu decoding. Here we investigate the effect of two additional tRNA modification systems on RpoS translation; the analysis was also extended to another High UUX-leucine codon (HULC) protein, Host Factor for phage Qβ (Hfq). One tRNA modification, the addition of the 2’-O-methylcytidine/uridine 34 (C/U34m) tRNA modification by tRNA (cytidine/uridine-2’O)-ribose methyltransferase L (TrmL), requires the presence of the N6-isopentenyladenosine 37 (i6A37) and therefore it seemed possible that the defect in RpoS translation in the absence of i6A37 prenyl transferase (MiaA) was in fact due to the inability to add the C/U34m modification to UUX-Leu tRNAs. The second modification, addition of 2-thiouridine (s2U), part of (mnm5s2U34), is dependent on tRNA 2-thiouridine synthesizing protein A (TusA), previously shown to affect RpoS levels. We compared expression of PBAD-rpoS990-lacZ translational fusions carrying wild-type UUX leucine codons with derivatives in which UUX codons were changed to CUX codons, in the presence and absence of TrmL or TusA. The absence of these proteins, and therefore presumably the modifications they catalyze, both abolished PBAD-rpoS990-lacZ translation activity. UUX-Leu to CUX-Leu codon mutations in rpoS suppressed the trmL requirement for PBAD-rpoS990-lacZ expression. Thus, it is likely that the C/U34m and s2U34 tRNA modifications are necessary for full rpoS translation. We also measured PBAD-hfq306-lacZ translational fusion activity in the absence of C/U34m (trmL) or i6A37 (miaA). The absence of i6A37 resulted in decreased PBAD-hfq306-lacZ expression, consistent with a role for i6A37 tRNA modification for hfq translation.

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“…A recent study in E. coli suggested context-dependent effects of loss of MiaA on efficient translation of rpoS mRNA [29,37]. Steady-state levels of RpoD and RpoS were compared by Western blot analysis and showed a two to threefold decrease in the steady-state levels of RpoS, but not RpoD, in the E. coli miaA mutant [29].…”

Section: Discussionmentioning

confidence: 98%

“…Recently, it was suggested that tRNA modification may regulate bacterial physiology via effects on global transcriptional regulators such as RpoS, based on observations in E. coli that MiaA was required for robust expression of RpoS [29]. The effect may be due to the high proportion of MiaA-dependent codons in the RpoS coding sequence, resulting in reduced translation and levels of that sigma factor [29,37].…”

Section: Introductionmentioning

confidence: 99%

Disruption of MiaA provides insights into the regulation of phenazine biosynthesis under suboptimal growth conditions in Pseudomonas chlororaphis 30-84

Wang

Pierson

et al. 2017

Microbiology

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Many products of secondary metabolism are activated by quorum sensing (QS), yet even at cell densities sufficient for QS, their production may be repressed under suboptimal growth conditions via mechanisms that still require elucidation. For many beneficial plant-associated bacteria, secondary metabolites such as phenazines are important for their competitive survival and plant-protective activities. Previous work established that phenazine biosynthesis in Pseudomonas chlororaphis 30-84 is regulated by the PhzR/PhzI QS system, which in turn is regulated by transcriptional regulator Pip, two-component system RpeA/RpeB and stationary phase/stress sigma factor RpoS. Disruption of MiaA, a tRNA modification enzyme, altered primary metabolism and growth leading to widespread effects on secondary metabolism, including reduced phenazine production and oxidative stress tolerance. Thus, the miaA mutant provided the opportunity to examine the regulation of phenazine production in response to altered metabolism and growth or stress tolerance. Despite the importance of MiaA for translation efficiency, the most significant effect of miaA disruption on phenazine production was the reduction in the transcription of phzR, phzI and pip, whereas neither the transcription nor translation of RpeB, a transcriptional regulator of pip, was affected. Constitutive expression of rpeB or pip in the miaA mutant completely restored phenazine production, but it resulted in further growth impairment. Constitutive expression of RpoS alleviated sensitivity to oxidative stress resulting from RpoS translation inefficiency in the miaA mutant, but it did not restore phenazine production. Our results support the model that cells curtail phenazine biosynthesis under suboptimal growth conditions via RpeB/Pip-mediated regulation of QS.

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The i⁶A37 tRNA modification is essential for proper decoding of UUX-Leucine codons during rpoS and iraP translation

Cited by 31 publications

References 63 publications

Case for the genetic code as a triplet of triplets

Case for the genetic code as a triplet of triplets

TrmL and TusA Are Necessary for rpoS and MiaA Is Required for hfq Expression in Escherichia coli

Disruption of MiaA provides insights into the regulation of phenazine biosynthesis under suboptimal growth conditions in Pseudomonas chlororaphis 30-84

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