title: Autoregulation of a bacterial sigma factor explored by using segmental isotopic labeling and NMR.
abstract: Bacterial sigma factors combine with the catalytic core RNA polymerase to direct the process of transcription initiation through sequence-specific interactions with the -10 and -35 elements of promoter DNA. In the absence of core RNA polymerase, the DNA-binding function of sigma is autoinhibited by its own N-terminal 90 amino acids (region 1.1), putatively by a direct interaction with conserved region 4.2, which binds the -35 promoter element. In the present work, this mechanism of autoinhibition was studied by using a combination of NMR spectroscopy and segmental isotopic labeling of a sigma70-like subunit from Thermotoga maritima. Our data argue strongly against a high-affinity interaction between these two domains. Instead we suggest that autoinhibition of DNA binding occurs through an indirect steric and/or electrostatic mechanism. More generally, the present work illustrates the power of segmental isotopic labeling for probing molecular interactions in large proteins by NMR.
authors.affiliation: Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, NY 10021, USA.
authors.forename: Julio A
authors.initials: JA
authors.lastname: Camarero
authors.forename: Alexander
authors.initials: A
authors.lastname: Shekhtman
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Campbell
authors.forename: Mark
authors.initials: M
authors.lastname: Chlenov
authors.forename: Tanja M
authors.initials: TM
authors.lastname: Gruber
authors.forename: Donald A
authors.initials: DA
authors.lastname: Bryant
authors.forename: Seth A
authors.initials: SA
authors.lastname: Darst
authors.forename: David
authors.initials: D
authors.lastname: Cowburn
authors.forename: Tom W
authors.initials: TW
authors.lastname: Muir
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D007554:Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020124:Thermotoga maritima / Q000378:metabolism*
chemical_list: D007554:Isotopes; D012808:Sigma Factor; D004247:DNA