Cross-linking of Escherichia coli RNA polymerase subunits: identification of beta' as the binding site of omega.
The omega protein is a peptide found in near-stoichiometric levels in highly purified Escherichia coli RNA polymerase. In order to determine the binding site of omega to RNA polymerase, we cross-linked omega to RNA polymerase with the hetero-bifunctional cross-linker N-hydroxysuccinimidyl 4-azidobenzoate and analyzed for cross-linked partners using antibodies raised against each of the subunits. Our analysis indicates that omega cross-links predominantly with the beta' subunit, while a very low level of cross-linking was detected to the alpha subunit. We did not detect cross-linking to either the sigma 70 or the beta subunits. This report demonstrates the utility of combining cross-linking and immunological techniques to determine interactions between RNA polymerase subunits.
Gentry
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Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Burgess
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D001386:Azides; D001665:Binding Sites; D015153:Blotting, Western; D003432:Cross-Linking Reagents; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D014466:Ultraviolet Rays
D001386:Azides; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; C029534:hydroxysuccinimidyl-4-azidobenzoate; D012321:DNA-Directed RNA Polymerases