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

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

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1
Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, NY 10021, USA.

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.

PMID:
12084914
PMCID:
PMC124302
DOI:
10.1073/pnas.132033899
[Indexed for MEDLINE]
Free PMC Article
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