Dissecting transcription regulatory pathways through a new bacterial one-hybrid reporter system

Abstract

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 ;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.

DarR, a TetR-like Transcriptional Factor, Is a Cyclic Di-AMP-responsive Repressor in Mycobacterium smegmatis

“…Bacterial One-hybrid Assays-The regulatory sequence of the darR gene was cloned into the reporter vector pBXcmT (20). DarR was fused into the N-terminal domain of the ␣-subunit of RNA polymerase in the pTRG vector (Stratagene).…”

“…DarR was fused into the N-terminal domain of the ␣-subunit of RNA polymerase in the pTRG vector (Stratagene). Bacterial one-hybrid assays were carried out as described previously (20).…”

Zebrafish IRF1 Regulates IFN Antiviral Response through Binding to IFNϕ1 and IFNϕ3 Promoters Downstream of MyD88 Signaling

“…This system allows identification of transcription factors (target) that exhibit the potential to interact with specific promoter DNA sequences (bait) in the reporter vector (Fig. 1A), because this interaction would stabilize the binding of RNAP, whereby activating the transcription of the HIS3 and aadA reporter gene (31). Initially, the feasibility of this system was verified by C. auratus IRF (CaIRF)3, which upregulates the expression of CaIFN, likely by binding to two ISRE/IRF-E motifs (ISRE1 and ISRE2) of the CaIFN promoter (19).…”

A TetR Family Transcriptional Factor Directly Regulates the Expression of a 3-Methyladenine DNA Glycosylase and Physically Interacts with the Enzyme to Stimulate Its Base Excision Activity in Mycobacterium bovis BCG

“…1, when the reporter plasmid containing either the upstream sequence of the mbaag gene (pBX-mbaagp) or bcg0878c promoter (pBXbcg0878cp) was co-transformed with the bait plasmid containing the BCG0878c regulator (pTRG-bcg0878c) into the E. coli reporter strain, the co-transformants grew very well on the screening medium. Positive co-transformants with pTRGRv3133c/pBX-Rv2031p, selected based on a previously reported interaction (17), also grew well on the medium. By contrast, no growth was observed for their corresponding selfactivated controls or the reported negative control composed of co-transformants with pTRG-Rv3133c⌬C/pBX-Rv2031p (19).…”

“…Bacterial One-hybrid Assay-A bacterial one-hybrid reporter system, which has been described previously (17), was used to examine interactions between the transcriptional factor and promoter. The promoter was cloned into pBXcmT, which is a derivative of the bacterial two-hybrid pBT bait plasmid (Invitrogen), whereas the transcription factor was cloned into pTRG (Invitrogen).…”