Identification of an N-terminal region of sigma 54 required for enhancer responsiveness.
Sigma 54 associates with bacterial core RNA polymerase and converts it into an enhancer-responsive enzyme. Deletion of the N-terminal 40 amino acids is known to result in loss of the ability to respond to enhancer binding proteins. In this work PCR mutagenesis and genetic screens were used to identify a small patch, from amino acids 33 to 37, that is required for proper response to activator in vivo. Site-directed single point mutants within this segment were constructed and studied. Two of these were defective in responding to the enhancer binding protein in vitro. The mutants could still direct the polymerase to bind to DNA and initiate transient melting. However, they failed in directing activator-dependent formation of a heparin-stable open complex. Thus, amino acid region 33 to 37 includes critical activation response determinants. This region overlaps the larger leucine patch negative-control region, suggesting that anti-inhibition and positive activation are closely coupled events.
Syed
A
A
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095-1569, USA.
Gralla
J D
JD
D000595:Amino Acid Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000235:genetics*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54