Abstract
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.
Highlights
Escherichia coli RNA Polymerase sigma S-subunit (RpoS) is an alternative sigma factor that facilitates physiological adaptation to general starvation and stationary phase growth [1,2]
Host Factor for phage Qβ (Hfq) was an attractive candidate, due to its elevated UUX-Leu codon usage ratio (Table S7, [19]) and its phylogenetically conserved cotranscription with miaA. We demonstrate that both tRNA (cytidine/uridine-2’O)-ribose methyltransferase L (TrmL) and thiouridine synthesizing protein A (TusA) are necessary for full RpoS translation and MiaA-catalyzed-i6 adenine 37 (A37) is necessary for hfq expression
In order to determine if mnm5 s2 U34 and C/U34m transfer RNA (tRNA) modifications may play a role in facilitating proper rpoS translation, we measured the effect of mutations in the enzymes necessary for these modifications on rpoS expression, using two different rpoS-lacZ translational fusion strains
Summary
Escherichia coli RpoS (σS ) is an alternative sigma factor that facilitates physiological adaptation to general starvation and stationary phase growth [1,2]. C/U34mmodification modification of these leucine tRNAs isoacceptors requires i6A37 modification [21] Taken together, this suggests that miaA and trmL may work together to affect rpoS expression and we investigated the role the trmL in rpoS translation in this work. I6 A37 modification [21] There is an elevated UUX-Leu to CUX-Leu ratio in the hfq open reading frame, characterizing it as a HULC protein that may, like RpoS, be sensitive to the i6 A37 tRNA modification [19]. We further tested our previous predictions on the role of the i6 A37 modification on expression of proteins with High UUX leucine Codon (HULC) content, using hfq as a model gene. We demonstrate that both TrmL and TusA are necessary for full RpoS translation and MiaA-catalyzed-i6 A37 is necessary for hfq expression
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