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J Biol Chem, Vol. 275, Issue 2, 1119-1127, January 14, 2000

Transcription Activation Mediated by the Carboxyl-terminal Domain of the RNA Polymerase -Subunit
MULTIPOINT MONITORING USING A FLUORESCENT PROBE^*

Olga N. Ozoline^§, Nobuyuki Fujita, and Akira Ishihama^¶

From the  Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan and the ^§ Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russian Federation

Conformational changes within the carboxyl-terminal domain of the Escherichia coli RNA polymerase -subunit (-CTD) upon interaction with the DNA UP element or the transcription factor cAMP receptor protein (CRP) were studied by monitoring the spectral parameters of a fluorescent dye, fluorescein mercuric acetate, conjugated to various positions of -CTD. When fluorescein mercuric acetate was conjugated to Cys located on helix I and the loop between helices III and IV, the spectral changes typical for DNA interaction were observed for the RNA polymerase-promoter binary complex with UP element-dependent rrnBP1 and the ternary complex with the CRP-dependent uxuAB promoter in the presence of cAMP/CRP. Likewise, the chemical nuclease iron-(p-bromoacetamidobenzyl)-EDTA conjugated to Cys-269 or Cys-272 introduced CRP-dependent cleavage of the uxuAB promoter, as in the case of rrnBP1 (Murakami, K., Owens, J. T., Belyaeva, T. A., Meares, C. F., Busby, S. J. W., and Ishihama, A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11274-11278), indicating that CRP rearranges the topology of the DNA contact surface in -CTD. Conformational changes in -CTD were also observed upon formation of a binary complex with the uxuAB (in the absence of CRP) and factor-independent T7D promoters. The spectral changes suggested that helix IV of -CTD approaches the negatively charged phosphate moiety of DNA. In agreement with this prediction, iron-(p-bromoacetamidobenzyl)-EDTA conjugated to Cys-309 induced extensive DNA cleavage upstream from the uxuAB promoter 35 element. We propose that helix IV of -CTD is involved in direct interaction with some promoters.

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