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

Julio A. Camarero, Alexander Shekhtman, Elizabeth A. Campbell, Mark Chlenov, Tanja M. Gruber, Donald A. Bryant, Seth A. Darst, David Cowburn, Tom W. Muir

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100 Scopus citations

Abstract

Bacterial σ 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 σ 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 σ70-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.

Original languageEnglish (US)
Pages (from-to)8536-8541
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume99
Issue number13
DOIs
StatePublished - Jun 25 2002

ASJC Scopus subject areas

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    Camarero, J. A., Shekhtman, A., Campbell, E. A., Chlenov, M., Gruber, T. M., Bryant, D. A., Darst, S. A., Cowburn, D., & Muir, T. W. (2002). Autoregulation of a bacterial σ factor explored by using segmental isotopic labeling and NMR. Proceedings of the National Academy of Sciences of the United States of America, 99(13), 8536-8541. https://doi.org/10.1073/pnas.132033899