The domain structure of sigma 54 as determined by analysis of a set of deletion mutants.
Escherichia coli sigma 54 was analyzed by making a series of 16 internal deletions within its gene and analyzing the properties of the mutant proteins. All of the mutant proteins except one were strongly defective in a growth test that relied on sigma 54 function. Additional assays were applied to determine the causes of these defects. The assays monitored the following properties: the level of protein expression; ability to bind to the -24 promoter element of the glnAP2 promoter in vivo; the ability to bind to the -12 promoter element in vivo; ability to melt the promoter start site in vivo; ability to bind the Rhizobium meliloti nifH promoter in vitro; and the ability to form a sigma 54-core RNA polymerase complex (E sigma 54 holoenzyme) in vitro. The analysis shows a modular structure in that certain regions of the protein predominate in contributing to each of these properties. A large carboxyl region of the protein is essential for promoter binding. A smaller amino-terminal segment is essential for DNA melting. An element essential for the forming the E sigma 54 holoenzyme lies between these two regions. None of these domains resemble those of sigma 70 and this difference is discussed in view of the different transcription mechanisms directed by the two proteins.
Wong
C
C
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024-1569.
Tintut
Y
Y
Gralla
J D
JD
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D009586:Nitrogen Fixation / Q000235:genetics; D009591:Nitrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010088:Oxidoreductases; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016962:Sinorhizobium meliloti / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012808:Sigma Factor; C497751:rpoN protein, E coli; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54