Functional Analysis of Promoters, mRNA Cleavage, and mRNA Secondary Structure on esxB-esxA in Mycolicibacterium smegmatis

The ESX-1 secretion system is critical for the virulence of as well as for conjugation in the saprophytic model . EsxB (CFP-10) and EsxA (ESAT-6) are secreted effectors required for the function of ESX-1 systems. While some transcription factors regulating the expression of and have been identified, little work has addressed their promoter structures or other determinants of their expression. Here, we defined two promoters, one located two genes upstream of and one located immediately upstream, that contribute substantially to the expression of and . We also defined an mRNA cleavage site within the 5' untranslated region (UTR) and found that a single-nucleotide substitution reprogramed the position of this cleavage event without impacting - transcript abundance. We furthermore investigated the impact of a double stem-loop structure in the 5' UTR and found that it does not confer stability on a reporter gene transcript. Consistent with this, there was no detectable correlation between mRNA half-life and secondary structure near the 5' ends of 5' UTRs on a transcriptome-wide basis. Collectively, these data shed light on the determinants of expression in as well as provide broader insight into the determinants of mRNA cleavage in mycobacteria and the relationship between 5' UTR secondary structure and mRNA stability.