Monitoring of RNA polymerase−DNA UP element interaction by a fluorescent probe conjugated to α subunit

The carboxy‐terminal domain (CTD) of Escherichia coli RNA polymerase α 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 rrnB P1, trp P and lac P2 promoters but not with many other promoters including mutant rrnB P1 without the UP element, supporting the idea that Cys269 or the domain including Cys269 is involved in UP element recognition. Both trp P and lac P2 have sequence similarity to the rrnB P1 UP element. The chemical modification of RNA polymerase, however, did not affect an apparent equilibrium dissociation constant with rrnB P1, as measured by gel‐retardation assays, indicating that the DNA‐binding ability is retained even after FMMA conjugation. Interaction with the rrnB P1 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 αCTD 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 trp P, supporting the prediction that this promoter carries an rrnB P1‐type UP element.