Transcription activation at Escherichia coli promoters dependent on the cyclic AMP receptor protein: effects of binding sequences for the RNA polymerase alpha-subunit.
Transcription activation at two semi-synthetic Escherichia coli promoters, CC(-41.5) and CC(-72.5), is dependent on the cyclic AMP receptor protein (CRP) that binds to sites centred 41.5 and 72.5 bp upstream from the respective transcription startpoints. An UP-element that can bind the C-terminal domain of the RNA polymerase (RNAP) alpha-subunit was cloned upstream of the DNA site for CRP at CC(-41.5) and downstream of the DNA site for CRP at CC(-72.5). In both cases CRP-dependent promoter activity was increased by the UP-element, but CRP-independent activity was not increased. DNase I footprinting was exploited to investigate the juxtaposition of bound CRP and RNAP alpha-subunits. In both cases, CRP and RNAP alpha-subunits occupy their cognate binding sites in ternary CRP-RNAP promoter complexes. RNAP alpha-subunits can occupy the UP-element in the absence of CRP, but this is not sufficient for open complex formation. The positive effects of binding RNAP alpha-subunits upstream of the DNA site for CRP at -41.5 are suppressed if the UP-element is incorrectly positioned.
Savery
N J
NJ
School of Biochemistry, University of Birmingham, U.K.
Rhodius
V A
VA
Wing
H J
HJ
Busby
S J
SJ
D001483:Base Sequence; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015533:Transcriptional Activation
D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases