Monitoring of RNA polymerase-DNA UP element interaction by a fluorescent probe conjugated to alpha subunit.
The carboxy-terminal domain (CTD) of Escherichia coli RNA polymerase alpha subunit was specifically modified by a reporter label, fluorescein mercuric acetate (FMMA), conjugated to Cys269 on the surface of UP element recognition helix. The modified enzyme was used to investigate RNA polymerase interaction with different promoters, either with or without an UP element. In a single-round transcription assay, the activity of modified RNA polymerase was found to decrease as measured with rrnBP1, trpP and lacP2 promoters but not with many other promoters including mutant rrnBP1 without the UP element, supporting the idea that Cys269 or the domain including Cys269 is involved in UP element recognition. Both trpP and lacP2 have sequence similarity to the rrnBP1 UP element. The chemical modification of RNA polymerase, however, did not affect an apparent equilibrium dissociation constant with rrnBP1, as measured by gel-retardation assays, indicating that the DNA-binding ability is retained even after FMMA conjugation. Interaction with the rrnBP1 UP element led to substantial alterations in the spectral parameters of the reporter label, which are different from those induced by complex formation with promoters without UP elements. A pronounced spectral blue shift suggests that the labeled surface of alphaCTD closely approaches the charged UP DNA helix. These observations imply that the fluorescent labeling at Cys269 can be used as a good tool for monitoring the presence or absence of an UP element in a given promoter. Spectral parameters of the label displayed the spectral blue shift when the modified RNA polymerase interacted with trpP, supporting the prediction that this promoter carries an rrnBP1-type UP element.
Ozoline
O N
ON
National Institute of Genetics, Mishima, Shizuoka, Japan.
Fujita
N
N
Murakami
K
K
Ishihama
A
A
D001483:Base Sequence; D001665:Binding Sites; D015220:Calcium Channels / Q000235:genetics; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005456:Fluorescent Dyes; D005798:Genes, Bacterial / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D016415:Sequence Alignment; D013057:Spectrum Analysis; D050052:TRPC Cation Channels; D014158:Transcription, Genetic
D015220:Calcium Channels; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D012321:DNA-Directed RNA Polymerases