The Screening And Characterization Of New Transcription Factors Of Mycobacterium Tuberculosis

The interactions between transcription factors and target DNA have widespread biological significance in the regulation of gene expression. However, it is usually difficult to find transcription regulatory factors that recognize specific target promoters. It is very hard to extensively study the transcriptional regulation in human pathogen Mycobacterium tuberculosis, which grows extremely slow. Therefore, it is very important to develop a convenient and safe strategy for uncovering unique transcriptional regulation genes and new drug targets in this pathogen.In this study, we showed that a newly developed bacterial one-hybrid reporter vector system could provide a convenient and rapid strategy to determine the specific interaction between target DNA sequences and their transcription factors. Then, we have uncovered a number of new transcription factors that participate in the regulation of stress response and fatty acid metabolisms in M. tuberculosis. This study has got the following main results:①We have shown that the new B1H system can be used conveniently to determine the DNA-binding specificity of the transcription regulator Rv3133c to a previously reported promoter region of the gene Rv2031 in Mycobacterium tuberculosis. And we have also determined the screening intensity of this system.②The new B1H system was shown to be also used to successfully determine the DNA-protein interactions between the replication initiation protein and replication origin Oril.③We have uncovered the new transcription factors for regulating the expressions of M. tuberculosis genes Rv2031 and Rv3874/3875.④We have screened more than 20 promoter regions of M. tuberculosis genes using this new system, and about 100 new transcription factors were found to participate in the regulation of stress response and fatty acid metabolisms.⑤WhiB3, a WhiB-like transcriptional factor, was identified for the first time to bind with the promoter sequences of most stress response and fatty acid metabolism genes, and the potential binding site of WhiB3 was located in a non-conserved 60bp region.Therefore, we have proved that the new bacterial one-hybrid reporter vector system can successfully detect and uncover new transcription factors. And these results have offered important data in the dissection of the transcription regulations in M. tuberculosis. And the strategy should be applicable in the study of DNA-protein interactions in a wide range of biological organisms.