Structure-function studies of Escherichia coli RpoH (sigma32) by in vitro linker insertion mutagenesis.
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.
Narberhaus
Franz
F
Institut für Mikrobiologie, Eidgenössische Technische Hochschule, CH-8092 Zürich, Switzerland. fnarber@micro.biol.ethz.ch
Balsiger
Sylvia
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D018833:Chaperonins; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D007763:Lac Operon; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C069774:heat-shock sigma factor 32; D018833:Chaperonins