title: Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA.
abstract: 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. Our combined data lead to a better understanding of the mechanism of RbpA function as a transcription activator.
authors.affiliation: Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065;
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Hubin
authors.affiliation: School of Life Sciences, University of Sussex, Brighton BN1 9QG, United Kingdom;
authors.forename: Aline
authors.initials: A
authors.lastname: Tabib-Salazar
authors.affiliation: School of Life Sciences, University of Sussex, Brighton BN1 9QG, United Kingdom;
authors.forename: Laurence J
authors.initials: LJ
authors.lastname: Humphrey
authors.affiliation: Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065;
authors.forename: Joshua E
authors.initials: JE
authors.lastname: Flack
authors.affiliation: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10065.
authors.forename: Paul Dominic B
authors.initials: PD
authors.lastname: Olinares
authors.affiliation: Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065;
authors.forename: Seth A
authors.initials: SA
authors.lastname: Darst
authors.affiliation: Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065; elizabeth.campbell0@gmail.com M.Paget@sussex.ac.uk.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Campbell
authors.affiliation: School of Life Sciences, University of Sussex, Brighton BN1 9QG, United Kingdom; elizabeth.campbell0@gmail.com M.Paget@sussex.ac.uk.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Paget
mesh_terms: D039903:Actinobacteria / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D014157:Transcription Factors
keywords: RbpA; X-ray crystallography; actinobacteria; mycobacteria; transcription