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

The translation of rpoS(σ(S)), the general stress/stationary phase sigma factor, is tightly regulated at the post-transcriptional level by several factors via mechanisms that are not clearly defined. One of these factors is MiaA, the enzyme necessary for the first step in theN(6)-isopentyl-2-thiomethyl adenosinemethyl adenosine 37 (ms(2)i(6)A37) tRNA modification. We tested the hypothesis that an elevated UUX-Leucine/total leucine codon ratio can be used to identify transcripts whose translation would be sensitive to loss of the MiaA-dependent modification. We identified iraPas another candidate MiaA-sensitive gene, based on the UUX-Leucine/total leucine codon ratio. AniraP-lacZ fusion was significantly decreased in the abse nce of MiaA, consistent with our predictive model. To determine the role of MiaA in UUX-Leucine decoding in rpoS and iraP, we measured β-galactosidase-specific activity of miaA(-)rpo Sandira P translational fusions upon overexpression of leucine tRNAs. We observed suppression of the MiaA effect on rpoS, and notira P, via overexpression of tRNA(LeuX)but not tRNA(LeuZ) We also tested the hypothesis that the MiaA requirement for rpoS and iraP translation is due to decoding of UUX-Leucine codons within the rpoS and iraP transcripts, respectively. We observed a partial suppression of the MiaA requirement for rpoS and iraP translational fusions containing one or both UUX-Leucine codons removed. Taken together, this suggests that MiaA is necessary for rpoS and iraP translation through proper decoding of UUX-Leucine codons and that rpoS and iraP mRNAs are both modification tunable transcripts (MoTTs) via the presence of the modification.