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

Olga N. Ozoline, Nobuyuki Fujita, Katsuhiko Murakami, Akira Ishihama

Research output: Contribution to journalArticle

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Abstract

The carboxy-terminal domain (CTD) of Escherichia coli RNA polymerase a 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 aCTD 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.

Original languageEnglish (US)
Pages (from-to)371-381
Number of pages11
JournalEuropean Journal of Biochemistry
Volume253
Issue number2
DOIs
StatePublished - Apr 15 1998

Fingerprint

DNA-Directed RNA Polymerases
Fluorescent Dyes
Monitoring
DNA
Labels
Assays
Chemical modification
Electrophoretic Mobility Shift Assay
Transcription
Labeling
Escherichia coli
Gels
Enzymes

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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abstract = "The carboxy-terminal domain (CTD) of Escherichia coli RNA polymerase a 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 aCTD 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.",
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Monitoring of RNA polymerase-DNA UP element interaction by a fluorescent probe conjugated to α subunit. / Ozoline, Olga N.; Fujita, Nobuyuki; Murakami, Katsuhiko; Ishihama, Akira.

In: European Journal of Biochemistry, Vol. 253, No. 2, 15.04.1998, p. 371-381.

Research output: Contribution to journalArticle

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