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Journal of Bacteriology, May 2003, p. 2731-2738, Vol. 185, No. 9
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.9.2731-2738.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Structure-Function Studies of Escherichia coli RpoH ({sigma}32) by In Vitro Linker Insertion Mutagenesis

Franz Narberhaus* and Sylvia Balsiger

Institut für Mikrobiologie, Eidgenössische Technische Hochschule, CH-8092 Zürich, Switzerland

Received 9 December 2002/ Accepted 20 February 2003

The sigma factor RpoH ({sigma}32) is the key regulator of the heat shock response in Escherichia coli. Many structural and functional properties of the sigma factor are poorly understood. To gain further insight into RpoH regions that are either important or dispensable for its cellular activity, we generated a collection of tetrapeptide insertion variants by a recently established in vitro linker insertion mutagenesis technique. Thirty-one distinct insertions were obtained, and their sigma factor activity was analyzed by using a groE-lacZ reporter fusion in an rpoH-negative background. Our study provides a map of permissive sites which tolerate linker insertions and of functionally important regions at which a linker insertion impairs sigma factor activity. Selected linker insertion mutants will be discussed in the light of known sigma factor properties and in relation to a modeled structure of an RpoH fragment containing region 2.

* Corresponding author. Mailing address: Institut für Mikrobiologie, ETH-Zentrum, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland. Phone: 41-1-632-2586. Fax: 41-1-632-1148. E-mail: fnarber{at}micro.biol.ethz.ch.

Journal of Bacteriology, May 2003, p. 2731-2738, Vol. 185, No. 9
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.9.2731-2738.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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