Studies on the functional topography of <i>Escherichia coli</i> RNA polymerase

Abstract

RNA polymerase was treated in the presence of promoter-containing templates with 16 affinity reagents, derivatives on NMPs, NDPs and NTPs with reactive substituents at the terminal phosphate. This treatment was followed by addition of a pyrimidine [alpha-32P]NTP. Due to 'catalytic competence' of some of the residues of the affinity reagents bound covalently near the active center at the first stage, active-center-catalyzed synthesis of a phosphodiester bond occurred, and radioactive residues with the general formula -pNpN (where p = radioactive phosphate) appeared covalently attached to the enzyme. Such affinity labelling was super-selective because affinity reagent residues bound outside the active center were not elongated and thus remained non-radioactive. Labelling took place only when the combination of the reagent and [alpha-32P]NTP corresponded to the sequence of nucleotides of the promoter. With reagents having short 'arms', only the beta subunit was labelled; the targets were His and/or Lys residues. With reagents having longer 'arms', the sigma subunit was also labelled.