S Unniraman1, V Nagaraja. 1. Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore-560012, India.
Abstract
BACKGROUND: The topological state of DNA is a result of the diverse influences of various topoisomerases present in the cell. Amongst these, DNA gyrase is the only enzyme that is capable of supercoiling DNA. In all the eubacterial cells tested so far, DNA gyrase has proved to be essential for survival. We have earlier cloned gyr genes from Mycobacterium smegmatis. Unlike the situation in Escherichia coli, genes encoding the two subunits of gyrase are present as a contiguous stretch in the M. smegmatis genome. RESULTS: We have demonstrated that the two subunits are encoded by a single dicistronic message, with the transcriptional start site mapping 57 base pairs upstream of the putative translational start of the gyrB ORF. The gyr promoter is specific to M. smegmatis and does not function in E. coli. We have shown that the synthesis of DNA gyrase in M. smegmatis is induced by novobiocin-a known inhibitor of gyrase. Short fragments encompassing the promoter region, when cloned in a promoter selection vector, do not show any response to changes in supercoil levels. Larger fragments show a supercoil sensitive behaviour, as seen in the genomic context. CONCLUSIONS: The gene structure and the transcriptional organization of the gyr operon suggest an overall regulatory scheme that is unique to mycobacteria. In contrast to E. coli, promoter and regions in its vicinity are not sufficient to confer supercoil sensitivity. Promoter distal regions- 600 bp downstream of the promoter-appear to be necessary for relaxation-stimulated transcription in M. smegmatis.
BACKGROUND: The topological state of DNA is a result of the diverse influences of various topoisomerases present in the cell. Amongst these, DNA gyrase is the only enzyme that is capable of supercoiling DNA. In all the eubacterial cells tested so far, DNA gyrase has proved to be essential for survival. We have earlier cloned gyr genes from Mycobacterium smegmatis. Unlike the situation in Escherichia coli, genes encoding the two subunits of gyrase are present as a contiguous stretch in the M. smegmatis genome. RESULTS: We have demonstrated that the two subunits are encoded by a single dicistronic message, with the transcriptional start site mapping 57 base pairs upstream of the putative translational start of the gyrB ORF. The gyr promoter is specific to M. smegmatis and does not function in E. coli. We have shown that the synthesis of DNA gyrase in M. smegmatis is induced by novobiocin-a known inhibitor of gyrase. Short fragments encompassing the promoter region, when cloned in a promoter selection vector, do not show any response to changes in supercoil levels. Larger fragments show a supercoil sensitive behaviour, as seen in the genomic context. CONCLUSIONS: The gene structure and the transcriptional organization of the gyr operon suggest an overall regulatory scheme that is unique to mycobacteria. In contrast to E. coli, promoter and regions in its vicinity are not sufficient to confer supercoil sensitivity. Promoter distal regions- 600 bp downstream of the promoter-appear to be necessary for relaxation-stimulated transcription in M. smegmatis.