ACTIVE SITE & MECHANISM OF RNA POLYMERASE

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The objective of this research is to provide new information about the function and catalytic mechanism of DNA-dependent RNA polymerase. To obtain such information, we propose to apply a variety of physical and chemical techniques, including absorption spectroscopy, circular dichroism (CD), fluorescence, stopped-flow kinetics, chemical and photochemical modification. These methods will be applied to RNA polymerase itself and to its complexes with DNA, substrates, substrate analogs and specific inhibitors. The aspects of RNA polymerase mechanism which we intend to explore are: (1) Binding of template to RNA polymerase; (2) The nature and location of catalytic groups in the enzyme; (3) The initiation and elongation process. 1. Binding of template. The nature of the conformational changes in the DNA and protein associated with formation of both closed and open promoter sites will be explored. Cloned restriction fragments of promoter-containing regions will be used. Fluorescence-labeled RNA polymerase will be used in stopped-flow kinetics studies of DNA binding to polymerase. The role of the Sigma subunit in DNA binding will be tested by determining whether Sigma might bind to single-stranded DNA. 2. Nature and location of catalytic groups. Chemical modification studies will be aimed at determining what groups participate in the catalysis. Nitration of tyrosine residues will be studied, especially those on the Sigma subunit. The site of affinity labels and photoaffinity labels will be determined in the sequence by chain separation cleavage and peptide mapping. Photooxidation with noncovalently and covalently attached dyes will be studied. 3. Initiation and elongation. The binding of NTPs and initiating dinucleotides to the enzyme and to enzyme-DNA complexes will be studied by spectroscopic methods. Specific promoter-containing fragments will be transcribed to a well-defined point to examine conformational differences between initiation and elongation complexes.