DNA-binding determinants of sigma 54 as deduced from libraries of mutations.
PCR mutagenesis was used to obtain libraries of mutations in the region between amino acids 300 and 400 in the DNA-binding domain of Escherichia coli sigma 54. Two hundred changes that did not alter function were identified. These were compared with a somewhat smaller number of changes that did alter function. Several important regions were identified. Single point mutations in two of these, near amino acids 363 and 383, destroyed the ability of sigma to bind DNA, as assayed by band shift analysis. A third segment from amino acids 327 to 347 is also a candidate for contributing to DNA binding. Comparison with data in the literature leads to testable proposals for the complex mode of DNA binding that is associated with sigma 54.
Guo
Y
Y
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, 90095-1569, USA.
Gralla
J D
JD
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D029968:Escherichia coli Proteins; D015723:Gene Library; D008345:Manganese; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012808:Sigma Factor; C497751:rpoN protein, E coli; D008345:Manganese; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54