Disulfide cross-linking indicates that FlgM-bound and free sigma28 adopt similar conformations.
The dissociable sigma subunit of bacterial RNA polymerase is required for the promoter-specific initiation of transcription. When bound to RNA polymerase, sigma makes sequence-specific promoter contacts and plays a crucial role in DNA melting. In isolation, however, sigma lacks significant promoter binding activity. In the crystal structure of the flagellar sigma factor, sigma(28), bound to the anti-sigma factor, FlgM, sigma(28) adopts a compact conformation in which the promoter binding surfaces are occluded by interdomain contacts. To test whether sigma(28) adopts this conformation in the absence of FlgM, we engineered a set of double cysteine mutants predicted to form interdomain disulfides in the conformation observed in the FlgM complex. We show that these disulfides form in both the presence and absence of FlgM. For two of the mutants, quantitative measurements of disulfide formation under equilibrium conditions suggest that the major solution conformation favors disulfide formation. The results indicate that the compact conformation of sigma(28) observed in the sigma(28)/FlgM structure is similar to the predominant conformation of free sigma(28) in solution. This finding suggests that autoinhibition of DNA binding in free sigma(28) is accomplished by steric occlusion of the promoter binding surfaces by interdomain interactions within the sigma factor as well as by a suboptimal distance between the promoter -10 and -35 element binding determinants in sigma(2) and sigma(4), respectively.
Sorenson
Margareta K
MK
The Rockefeller University, Box 224, 1230 York Avenue, New York, NY 10021, USA.
Darst
Seth A
SA
D019943:Amino Acid Substitution; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D008958:Models, Molecular; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; C064971:FliA protein, Bacteria; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D012808:Sigma Factor; C071266:FlgM protein, Bacteria; D003545:Cysteine