Post-Transcriptional Regulation of the MiaA Prenyl Transferase by CsrA and the Small RNA CsrB in Escherichia coli

MiaA is responsible for the addition of the isopentyl modification to adenine 37 in the anticodon stem loop of specific tRNAs in <i>Escherichia coli</i>. Mutants in <i>miaA</i> have pleotropic effects on the cell in <i>E. coli</i> and play a role in virulence gene regulation. In addition, MiaA is necessary for stress response gene expression by promoting efficient decoding of UUX-leucine codons, and genes with elevated UUX-leucine codons may be a regulatory target for i⁶A-modified tRNAs. Understanding the temporal nature of the i⁶A modification status of tRNAs would help us determine the regulatory potential of MiaA and its potential interplay with leucine codon frequency. In this work, we set out to uncover additional information about the synthesis of the MiaA. MiaA synthesis is primarily driven at the transcriptional level from multiple promoters in a complex operon. However, very little is known about the post-transcriptional regulation of MiaA, including the role of sRNAs in its synthesis. To determine the role of small RNAs (sRNAs) in the regulation of <i>miaA</i>, we constructed a chromosomal <i>miaA</i>-<i>lacZ</i> translational fusion driven by the arabinose-responsive P_BAD promoter and used it to screen against an <i>Escherichia coli</i> sRNA library (containing sRNAs driven by the IPTG-inducible P<i>_Lac</i> promoter). Our genetic screen and quantitative β-galactosidase assays identified CsrB and its cognate protein CsrA as potential regulators of <i>miaA</i> expression in <i>E. coli</i>. Consistent with our hypothesis that CsrA regulates <i>miaA</i> post-transcriptional gene expression through binding to the <i>miaA</i> mRNA 5' UTR, and CsrB binds and regulates <i>miaA</i> post-transcriptional gene expression through sequestration of CsrA levels, a deletion of <i>csrA</i> significantly reduced expression of the reporter fusion as well as reducing <i>miaA</i> mRNA levels. These results suggest that under conditions where CsrA is inhibited, <i>miaA</i> mRNA translation and thus MiaA-dependent tRNA modification may be limited.