tailieunhanh - Báo cáo khoa học: CmtR, a cadmium-sensing ArsR–SmtB repressor, cooperatively interacts with multiple operator sites to autorepress its transcription in Mycobacterium tuberculosis

CmtR is a repressor of the ArsR–SmtB family fromMycobacterium tuber-culosis that has been shown to sense Cd(II) and Pb(II) in Mycobacte-rium smegmatis. We establish here that CmtR binds cooperatively to multiple sites inM. tuberculosisDNA and protects an unusually long 90 bp AT-rich sequence from )80 bp to +10 with respect to its own initiation codon. | ễFEBS Journal CmtR a cadmium-sensing ArsR-SmtB repressor cooperatively interacts with multiple operator sites to autorepress its transcription in Mycobacterium tuberculosis Santosh Chauhan Anil Kumar Amit Singhal Jaya Sivaswami Tyagi and H. Krishna Prasad Department of Biotechnology All India Institute of MedicalSciences Ansari Nagar New Delhi India Keywords ArsR-SmtB repressor autoregulation CmtR metalloregulatory repressors Rv1994c Correspondence H. K. Prasad TB Immunology Laboratory Department of Biotechnology All India Institute of Medical Sciences New Delhi 110029 India Fax 91 11 26589286 Tel 91 11 26594994 E-mail hk_prasad@ J. S. Tyagi TB Molecular Biology Laboratory Department of Biotechnology All India Institute of MedicalSciences New Delhi 110029 India Fax 91 11 26588663 Tel 91 11 26588491 E-mail jstyagi@ CmtR is a repressor of the ArsR-SmtB family from Mycobacterium tuberculosis that has been shown to sense Cd II and Pb II in Mycobacterium smegmatis. We establish here that CmtR binds cooperatively to multiple sites in M. tuberculosis DNA and protects an unusually long 90 bp AT-rich sequence from -80 bp to 10 with respect to its own initiation codon. CmtR interacts with four hyphenated imperfect inverted repeats matching the consensus sequence TA GTAA-N45-TT GATA in the protected region. SDS-PAGE and formaldehyde crosslinking experiments showed that CmtR forms higher-order oligomers up to an octamer . The oligomerization of CmtR is in agreement with the cooperative binding of CmtR to multiple sites on DNA. Two promoters transcribe cmtR and the major promoter physically overlaps with CmtR binding sites. Autorepression of CmtR is mediated by cooperative interaction of CmtR with multiple sites on DNA that occlude the major operon promoter. The combined results of a GFP reporter assay an electromobility shift assay and a DNase I footprinting experiment establish that Cd II not Pb II disrupts the interaction of CmtR with DNA to de-repress