Abstract
The Escherichia coli genome encodes genes for seven different sigma subunit species while only having single genes for the alpha, beta, and beta' subunits that make up the RNA polymerase core enzyme. The various sigma factors compete for binding to the core enzyme, upon which they confer promoter DNA-specific transcription initiation to the polymerase. We have mapped a major interaction site between one of the sigma species, sigma70, and beta'. Using far-Western blotting analysis of chemically cleaved and genetically engineered protein fragments, we have identified a N-terminal fragment of beta' (residues 60-309) that could bind sigma70. We were able to more precisely map the interaction domain to amino acid residues 260-309 of beta' using nickel nitrilotriacetic acid co-immobilization assays.
Highlights
The RNA polymerase of Escherichia coli is a large, multisubunit enzyme existing in two forms
Elucidation of the structural characteristics of the core RNA polymerase- factor binding interaction will be very beneficial in fully understanding this aspect of regulation
A  subunit truncation, missing approximately 200 amino acids1 of the C terminus, was shown by glycerol gradient centrifugation to migrate with the other core subunits but was never seen in the -containing fractions (17)
Summary
NTCB Cleavage (33)—1 mg of inclusion body protein was resuspended in 1 ml of buffer B ϩ 8 M urea. The cleavage mixture was diluted 1:10 in buffer B ϩ 8 M urea and loaded onto a Ni2ϩ-NTA column as described above. Thermolysin Cleavage (35)—1 mg of inclusion body protein was resuspended in 100 l of buffer B ϩ 8 M urea and incubated for 15 min at 37 °C. Trypsin Cleavage (35)—1 mg of inclusion body protein was resuspended in 1 ml of buffer B ϩ 8 M urea and incubated for 15 min at 37 °C. The reaction mixture was loaded onto a Biospin-P6 column (Bio-Rad) pre-equilibrated with 1 ϫ kinase buffer and spun at 1,100 ϫ g for 4 min. Samples from the 70 flow-through, wash, and elution fractions were analyzed by SDS-PAGE
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