TY - JOUR
T1 - Autoregulation of a bacterial σ factor explored by using segmental isotopic labeling and NMR
AU - Camarero, Julio A.
AU - Shekhtman, Alexander
AU - Campbell, Elizabeth A.
AU - Chlenov, Mark
AU - Gruber, Tanja M.
AU - Bryant, Donald A.
AU - Darst, Seth A.
AU - Cowburn, David
AU - Muir, Tom W.
PY - 2002/6/25
Y1 - 2002/6/25
N2 - 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.
AB - 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.
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U2 - 10.1073/pnas.132033899
DO - 10.1073/pnas.132033899
M3 - Article
C2 - 12084914
AN - SCOPUS:0037173060
SN - 0027-8424
VL - 99
SP - 8536
EP - 8541
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 13
ER -