Autoregulation of a bacterial sigma factor explored by using segmental isotopic labeling and NMR

Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8536-41. doi: 10.1073/pnas.132033899.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • DNA
  • Isotopes
  • Nuclear Magnetic Resonance, Biomolecular
  • Promoter Regions, Genetic
  • Sigma Factor / chemistry
  • Sigma Factor / genetics
  • Sigma Factor / metabolism*
  • Thermotoga maritima / metabolism*

Substances

  • Isotopes
  • Sigma Factor
  • DNA