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Journal of Bacteriology, May 2001, p. 2866-2873, Vol. 183, No. 9
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.9.2866-2873.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Function-Based Selection and Characterization of Base-Pair Polymorphisms in a Promoter of Escherichia coli RNA Polymerase-sigma 70

Jian Xu,dagger Barbara C. McCabe, and Gerald B. Koudelka*

Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260-1300

Received 3 January 2001/Accepted 16 February 2001

We performed two sets of in vitro selections to dissect the role of the -10 base sequence in determining the rate and efficiency with which Escherichia coli RNA polymerase-sigma 70 forms stable complexes with a promoter. We identified sequences that (i) rapidly form heparin-resistant complexes with RNA polymerase or (ii) form heparin-resistant complexes at very low RNA polymerase concentrations. The sequences selected under the two conditions differ from each other and from the consensus -10 sequence. The selected promoters have the expected enhanced binding and kinetic properties and are functionally better than the consensus promoter sequence in directing RNA synthesis in vitro. Detailed analysis of the selected promoter functions shows that each step in this multistep pathway may have different sequence requirements, meaning that the sequence of a strong promoter does not contain the optimal sequence for each step but instead is a compromise sequence that allows all steps to proceed with minimal constraint.

* Corresponding author. Mailing address: Department of Biological Sciences, Cooke Hall, North Campus, State University of New York at Buffalo, Buffalo, NY 14260-1300. Phone: (716) 645-3489. Fax: (716) 645-2975. E-mail: koudelka{at}acsu.buffalo.edu.

dagger Present address: Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037-1099.

Journal of Bacteriology, May 2001, p. 2866-2873, Vol. 183, No. 9
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.9.2866-2873.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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