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 a lpha subunit was specifically modified by a reporter label, fluorescei n mercuric acetate (FMMA), conjugated to Cys269 on the surface of UP e lement recognition helix. The modified enzyme was used to investigate RNA polymerase interaction with different promoters, either with or wi thout an UP element. In a single-round transcription assay, the activi ty of modified RNA polymerase was found to decrease as measured with r rnBP1, trpP and lacP2 promoters but not with many other promoters incl uding mutant rrnBP1 without the UP element, supporting the idea that C ys269 or the domain including Cys269 is involved in UP element recogni tion. Both trpP and lacP2 have sequence similarity to the rrnBP1 UP el ement. The chemical modification of RNA polymerase, however, did nor a ffect an apparent equilibrium dissociation constant with rrnBP1, as me asured by gel-retardation assays, indicating that the DNA-binding abil ity is retained even after FMMA conjugation. Interaction with the rrnB P1 UP element led to substantial alterations in the spectral parameter s of the reporter label, which are different from those induced by com plex formation with promoters without UP elements. A pronounced spectr al blue shift suggests that the labeled surface of alpha CTD closely a pproaches the charged UP DNA helix. These observations imply that the fluorescent labeling at Cys269 can be used as a good tool for monitori ng the presence or absence of an UP element in a given promoter. Spect ral parameters of the label displayed the spectral blue shift when the modified RNA polymerase interacted with trpP, supporting the predicti on that this promoter carries an rrnBP1-type UP element.