Dissecting transcription regulatory pathways through a new bacterial one-hybrid reporter system.
Sequence-specific DNA-binding transcription factors have widespread biological significance in the regulation of gene expression. However, in lower prokaryotes and eukaryotic metazoans, it is usually difficult to find transcription regulatory factors that recognize specific target promoters. To address this, we have developed in this study a new bacterial one-hybrid reporter vector system that provides a convenient and rapid strategy to determine the specific interaction between target DNA sequences and their transcription factors. Using this system, we have successfully determined the DNA-binding specificity of the transcription regulator Rv3133c to a previously reported promoter region of the gene Rv2031 in Mycobacterium tuberculosis. In addition, we have tested more than 20 promoter regions of M. tuberculosis genes using this approach to determine if they interact with approximately 150 putative regulatory proteins. A variety of transcription factors are found to participate in the regulation of stress response and fatty acid metabolism, both of which comprise the core of in vivo-induced genes when M. tuberculosis invades macrophages. Interestingly, among the many new discovered potential transcription factors, the WhiB-like transcriptional factor WhiB3 was identified for the first time to bind with the promoter sequences of most in vivo-induced genes. Therefore, this study offers important data in the dissection of the transcription regulations in M. tuberculosis, and the strategy should be applicable in the study of DNA-binding factors in a wide range of biological organisms.
Guo
Manman
M
National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Feng
Hui
H
Zhang
Jun
J
Wang
Wenqin
W
Wang
Yi
Y
Li
Yuqing
Y
Gao
Chunhui
C
Chen
Huanchun
H
Feng
Ying
Y
He
Zheng-Guo
ZG
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000502:physiology*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D014157:Transcription Factors