1). If up to two mismatches were allowed, a further candidate CIRCE sequence was found upstream of cpn60.2, although two other potential matches were also found upstream of genes that are not usually part of the heat shock regulon (data not shown). It is thus likely that heat shock regulation of cpn10, cpn60.1 and cpn60.2 is mediated by the HrcA protein binding at CIRCE sequences, Panobinostat order but this remains to be proven. No CIRCE sequence was found upstream of cpn60.3, consistent with the observation that it is not induced by heat shock.
In M. tuberculosis, although cpn10 and cpn60.1 are adjacent on the chromosome, two putative transcriptional start sites have been proposed (Kong et al., 1993). One of these is upstream of cpn10, in the region containing the CIRCE sequence
that binds HrcA to regulate the heat shock response (Zuber & Schumann, 1994; Stewart et al., 2002). A second was identified 29 bp upstream of the cpn60.1 gene. However, a more recent report showed no promoter activity in this intergenic region (Aravindhan et al., 2009), raising the possibility that there is learn more a post-transcriptional cleavage of the mRNA for this operon. Because of this, and because our results showed that in M. smegmatis the adjacent cpn10 and cpn60.1 genes are expressed at significantly different levels under similar conditions, we used 5′RACE with the primers cpn60.1 gsp1, cpn60.1 gsp2 and cpn10 gsp1 to determine the transcriptional start sites of the cpn10 and cpn60.1 genes. The results showed two potential
transcriptional start sites, one 133 bp upstream from the cpn10 gene and the second in the intergenic region 31 bp upstream of the cpn60.1 gene (Fig. 1), similar to earlier findings with M. tuberculosis. To investigate whether the intergenic region did indeed contain a promoter, varying lengths of upstream regions of the chaperonin genes and the why cpn10–cpn60.1 intergenic region (Fig. 1) were cloned into the pSD5B reporter plasmid, and LacZ activity was measured following the transformation of these plasmids into M. smegmatis mc2155. Only the regions upstream of cpn10 and cpn60.2 exhibited promoter activity. Neither the shorter nor the longer intergenic fragment reported any promoter activity, as would have been expected had the putative start site identified shortly upstream of the cpn60.1 gene been genuine (Fig. 1). We therefore conclude that the mRNA 5′-end observed between cpn10 and cpn60.1 is likely to arise from a specific post-transcriptional cleavage event, similar to the situation reported in M. tuberculosis. The lower levels of expression of cpn60.1 compared with cpn10 may thus result from differential stabilities of the mRNAs for these two genes. This may have evolved from a need to match the levels of expression of the essential cpn10 and cpn60.2 genes, despite cpn10 being in an operon with the nonessential cpn60.1.