Open AccessAutoregulation of a bacterial σ factor explored by using segmental isotopic labeling and NMR
Author(s) -
Julio A. Camarero,
Alexander Shekhtman,
Elizabeth A. Campbell,
Mark Chlenov,
Tanja M. Gruber,
Donald A. Bryant,
Seth A. Darst,
David Cowburn,
Tom W. Muir
Publication year - 2002
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.132033899
Subject(s) - nuclear magnetic resonance spectroscopy , dna , biology , rna polymerase , biochemistry , sigma factor , polymerase , biophysics , protein subunit , chemistry , rna , stereochemistry , gene
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.