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 Escherichia 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, fluorescein mercuric acetate, conjugated to various positions of alpha-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 alpha-CTD. Conformational changes in alpha-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 alpha-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 alpha-CTD is involved in direct interaction with some promoters.
Ozoline
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ON
Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.
Fujita
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N
Ishihama
A
A
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D002373:Cyclic AMP Receptor Protein; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit