Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA

Hubin, Elizabeth A.; Tabib-Salazar, Aline; Humphrey, Laurence J.; Flack, Joshua E.; Olinares, Paul Dominic B.; Darst, Seth A.; Campbell, Elizabeth A.; Paget, Mark S.

doi:10.1073/pnas.1504942112

Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA

Journal Article · Tue May 26 00:00:00 EDT 2015 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.1504942112· OSTI ID:1229396

Hubin, Elizabeth A. ^[1]; Tabib-Salazar, Aline ^[2]; Humphrey, Laurence J. ^[3]; Flack, Joshua E. ^[4]; Olinares, Paul Dominic B. ^[1]; Darst, Seth A. ^[1]; Campbell, Elizabeth A. ^[1]; Paget, Mark S. ^[3]

Rockefeller Univ., New York, NY (United States)
Univ. of Sussex, Brighton (United Kingdom); Imperial College, London (United Kingdom)
Univ. of Sussex, Brighton (United Kingdom)
Rockefeller Univ., New York, NY (United States); Medical Research Council Lab. of Molecular Biology, Cambridge (United Kingdom)

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 σ-factors. To delineate the molecular mechanism of RbpA, we show that the Mtb RbpA σ-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 σ-factor, σ^A. The structure explains the basis of σ-selectivity by RbpA, showing that RbpA interacts with conserved regions of σ^A as well as the nonconserved region (NCR), which is present only in housekeeping σ-factors. Thus, the structure is the first, to our knowledge, to show a protein interacting with the NCR of a σ-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. As a result, our combined data lead to a better understanding of the mechanism of RbpA function as a transcription activator.

View Accepted Manuscript (DOE)

Research Organization:: Brookhaven National Laboratory (BNL), Upton, NY (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); NIH; Biotechnology and Biological Sciences Research Council

OSTI ID:: 1229396

Report Number(s):: BNL--111472-2015-JA

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 23 Vol. 112; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
RbpA
X-ray crystallography
actinobacteria
mycobacteria
transcription

Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA

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