Transcription activation mediated by the carboxyl-terminal domain of the RNA polymerase alpha-subunit - Multipoint monitoring using a fluorescent probe

Conformational changes within the carboxyl-terminal domain of the Escherich ia coli RNA polymerase alpha-subunit (alpha-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, fluore scein mercuric acetate, conjugated to various positions of alpha-CTD. When fluorescein mercuric acetate was conjugated to Cys located on helix I and t he loop between helices III and TV, the spectral changes typical for DNA in teraction 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 nucleas e iron-(p-bromoacetamidobenzyl)-EDTA conjugated to Cys-269 or Cys-272 intro duced CRP-dependent cleavage of the uxuAB promoter, as in the case of rrnBP 1 (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 i n alpha-CTD. Conformational changes in alpha-CTD were also observed upon fo rmation of a binary complex with the uxuAB (in the absence of CRP) and fact or-independent T7D promoters. The spectral changes suggested that helix TV of alpha-CTD approaches the negatively charged phosphate moiety of DNA, In agreement with this prediction, iron-(p-bromoacetamidobenzyl)-EDTA conjugat ed to Cys-309 induced extensive DNA cleavage upstream from the uxuAB promot er -35 element, We propose that helix IV of alpha-CTD is involved in direct interaction with some promoters.