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Journal of Bacteriology, May 2002, p. 2634-2641, Vol. 184, No. 10
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.10.2634-2641.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Using Disulfide Bond Engineering To Study Conformational Changes in the ß'260-309 Coiled-Coil Region of Escherichia coli RNA Polymerase during {sigma}70 Binding

Larry C. Anthony,1 Alan A. Dombkowski,2 and Richard R. Burgess1*

McArdle Laboratory for Cancer Research, University of Wisconsin—Madison, Madison, Wisconsin,1 Department of Pharmaceutical Chemistry, University of Michigan, Ann Arbor, Michigan2

Received 16 January 2002/ Accepted 25 February 2002

RNA polymerase of Escherichia coli is the sole enzyme responsible for mRNA synthesis in the cell. Upon binding of a sigma factor, the holoenzyme can direct transcription from specific promoter sequences. We have previously defined a region of the ß' subunit (ß'260-309, amino acids 260 to 309) which adopts a coiled-coil conformation shown to interact with {sigma}70 both in vitro and in vivo. However, it was not known if the coiled-coil conformation was maintained upon binding to {sigma}70. In this work, we engineered a disulfide bond within ß'240-309 that locks the ß' coiled-coil region in the coiled-coil conformation, and we show that this "locked" peptide is able to bind to {sigma}70. We also show that the locked coiled-coil is capable of inducing a conformational change within {sigma}70 that allows recognition of the -10 nontemplate strand of DNA. This suggests that the coiled-coil does not adopt a new conformation upon binding {sigma}70 or upon recognition of the -10 nontemplate strand of DNA.

* Corresponding author. Mailing address: McArdle Laboratory for Cancer Research, University of Wisconsin—Madison, 1400 University Ave., Madison, WI 53706-1599. Phone: (608) 263-2635. Fax: (608) 262-2824. E-mail: burgess{at}oncology.wisc.edu.

Journal of Bacteriology, May 2002, p. 2634-2641, Vol. 184, No. 10
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.10.2634-2641.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Sorenson, M. K., Darst, S. A. (2006). Disulfide cross-linking indicates that FlgM-bound and free {sigma}28 adopt similar conformations. Proc. Natl. Acad. Sci. USA 103: 16722-16727 [Abstract] [Full Text]  
  • Berghofer-Hochheimer, Y., Lu, C. Z., Gross, C. A. (2005). Altering the interaction between {sigma}70 and RNA polymerase generates complexes with distinct transcription-elongation properties. Proc. Natl. Acad. Sci. USA 102: 1157-1162 [Abstract] [Full Text]  
  • Mahren, S., Braun, V. (2003). The FecI Extracytoplasmic-Function Sigma Factor of Escherichia coli Interacts with the {beta}' Subunit of RNA Polymerase. J. Bacteriol. 185: 1796-1802 [Abstract] [Full Text]  
  • Anthony, L. C., Burgess, R. R. (2002). Conformational Flexibility in sigma 70 Region 2 during Transcription Initiation. J. Biol. Chem. 277: 46433-46441 [Abstract] [Full Text]  


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