期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：23
页码：7171-7176
DOI：10.1073/pnas.1504942112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceInitiation of transcription in bacteria relies on a multisubunit RNA polymerase in concert with a dissociable {sigma}-subunit that confers promoter recognition and opening to reveal the DNA template strand. RbpA, a transcription activator unique to Actinobacteria and essential in Mycobacterium tuberculosis, associates tightly with {sigma} and is required for efficient initiation, although its mechanism of action is unclear. Here, we solve the crystal structure of an M. tuberculosis {sigma}-RbpA complex and present evidence indicating that RbpA activates transcription through unexpected contacts with promoter DNA. The work sheds light on the mechanism of transcription initiation by M. tuberculosis RNA polymerase, which is a proven antibiotic target. Gene expression is highly regulated at the step of transcription initiation, and transcription activators play a critical role in this process. RbpA, an actinobacterial transcription activator that is essential in Mycobacterium tuberculosis (Mtb), binds selectively to group 1 and certain group 2 {sigma}-factors. To delineate the molecular mechanism of RbpA, we show that the Mtb RbpA {sigma}-interacting domain (SID) and basic linker are sufficient for transcription activation. We also present the crystal structure of the Mtb RbpA-SID in complex with domain 2 of the housekeeping {sigma}-factor, {sigma}A. The structure explains the basis of {sigma}-selectivity by RbpA, showing that RbpA interacts with conserved regions of {sigma}A as well as the nonconserved region (NCR), which is present only in housekeeping {sigma}-factors. Thus, the structure is the first, to our knowledge, to show a protein interacting with the NCR of a {sigma}-factor. We confirm the basis of selectivity and the observed interactions using mutagenesis and functional studies. In addition, the structure allows for a model of the RbpA-SID in the context of a transcription initiation complex. Unexpectedly, the structural modeling suggests that RbpA contacts the promoter DNA, and we present in vivo and in vitro studies supporting this finding. Our combined data lead to a better understanding of the mechanism of RbpA function as a transcription activator.
