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

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 N(6)-isopentenyladenosine 37 (i(6)A37) and therefore it seemed possible that the defect in RpoS translation in the absence of i(6)A37 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 (s(2)U), part of (mnm(5)s(2)U34), is dependent on tRNA 2-thiouridine synthesizing protein A (TusA), previously shown to affect RpoS levels. We compared expression of P(BAD)-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 P(BAD)-rpoS990-lacZ translation activity. UUX-Leu to CUX-Leu codon mutations in rpoS suppressed the trmL requirement for P(BAD)-rpoS990-lacZ expression. Thus, it is likely that the C/U34m and s(2)U34 tRNA modifications are necessary for full rpoS translation. We also measured P(BAD)-hfq306-lacZ translational fusion activity in the absence of C/U34m (trmL) or i(6)A37 (miaA). The absence of i(6)A37 resulted in decreased P(BAD)-hfq306-lacZ expression, consistent with a role for i(6)A37 tRNA modification for hfq translation.