467 Post-transcriptional regulation of the MiaA prenyl transferase by the small RNA CsrB in Escherichia coli (E. coli)

Abstract

OBJECTIVES/GOALS: MiaA is a highly conserved prenyl transferase that catalyzes synthesis of the i6A37 tRNA modification in E. coli. While transcriptional regulation of MiaA is well characterized, there is no information on the MiaA post-transcriptional regulation. The aim of this study is to characterize the post-transcriptional regulation of the MiaA gene in E. coli. METHODS/STUDY POPULATION: To characterize the post-transcriptional regulation of miaA, we executed a targeted genetic screen of an E. coli small RNA library on a miaA-lacZ translational reporter fusion strain to identify small RNAs (sRNAs) that modulate MiaA translation or transcription termination. We also measured MiaA mRNA levels and miaA-lacZ activity in the absence or over-expression of candidate sRNA regulators of MiaA. We also measured MiaA mRNA levels in the absence of RNaseE and PNPase, two enzymes involved in mRNA turnover. Finally, we measured the ability of purified recombinant CsrA to bind to the MiaA mRNA transcript in vitro. RESULTS/ANTICIPATED RESULTS: We identified the carbon sensing sRNA CsrB and its cognate protein interaction partner CsrA, as potential post-transcriptional regulators of MiaA. Over-expression of CsrB fully repressed miaA-lacZ activity and MiaA mRNA levels. The absence of CsrA resulted in a defective miaA-lacZ activity and a 10-fold decrease in MiaA mRNA levels. We also identified an increase in the MiaA mRNA half-life particularly in the absence of RNaseE. Our results demonstrate an additional layer of regulation for the miaA operon by the CsrA/CsrB protein-sRNA system. DISCUSSION/SIGNIFICANCE: MiaA is a highly conserved bacterial protein. Our data may represent phenomena in an array of bacteria that could be targeted by novel antibiotics. The human MiaA homologue, TRIT1, plays a role in mitochondrial disorders. We anticipate that information garnered from MiaA studies will elucidate TRIT1 function and its role in mitochondrial disorders.