Exploitation of a chemical nuclease to investigate the location and orientation of the Escherichia coli RNA polymerase alpha subunit C-terminal domains at simple promoters that are activated by cyclic AMP receptor protein.
The C-terminal domain of the alpha subunit (alphaCTD) of bacterial RNA polymerase plays an important role in promoter recognition. It is known that alphaCTD binds to the DNA minor groove at different locations at different promoters via a surface-exposed determinant, the 265 determinant. Here we describe experiments that permit us to determine the location and orientation of binding of alphaCTD at any promoter. In these experiments, a DNA cleavage reagent is attached to specific locations on opposite faces of the RNA polymerase alpha subunit. After incorporation of the tagged alpha subunits into holo-RNA polymerase, patterns of DNA cleavage due to the reagent are determined in open complexes. The locations of DNA cleavage due to the reagent attached at different positions allow the position and orientation of alphaCTD to be deduced. Here we present data from experiments with simple Escherichia coli promoters that are activated by the cyclic AMP receptor protein.
Lee
David J
DJ
School of Biosciences, the University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
Busby
Stephen J W
SJ
Lloyd
Georgina S
GS
D001483:Base Sequence; D003062:Codon; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics*; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D003062:Codon; D002373:Cyclic AMP Receptor Protein; D004247:DNA; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit; D003545:Cysteine