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 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 s²U34 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 i⁶A37 (miaA). The absence of i⁶A37 resulted in decreased P_BAD-hfq306-lacZ expression, consistent with a role for i⁶A37 tRNA modification for hfq translation.

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