Photoaffinity labeling of DNA-dependent RNA polymerase from Escherichia coli with 8-azidoadenosine 5'-triphosphate

Abstract

A photoaffinity analogue of adenosine 5'-triphosphate (ATP), 8-azidoadenosine 5'-triphosphate (8-N3ATP), has been used to elucidate the role of the various subunits involved in forming the active site of Escherichia coli DNA-dependent RNA polymerase. 8-N3ATP was found to be a competitive inhibitor of the enzyme with respect to the incorporation of ATP with Ki = 42 microM, while uridine 5'-triphosphate (UTP) incorporation was not affected. UV irradiation of the reaction mixture containing RNA polymerase and [gamma-32P]-8-N3ATP induced covalent incorporation of radioactive label into the enzyme. Analysis by gel filtration and nitrocellulose filter binding indicated specific binding. Subunit analysis by sodium dodecyl sulfate and sodium tetradecyl sulfate gel electrophoresis and autoradiography of the labeled enzyme showed that the major incorporation of radioactive label was in beta' and sigma, with minor incorporation in beta and alpha. The same pattern was observed in both the presence and absence of poly[d(A-T)] and poly[d(A-T)] plus ApU. Incorporation of radioactive label in all bands was significantly reduced by 100-150 microM ATP, while 100-200 microM UTP did not show a noticeable effect. Our results indicate major involvement of the beta' and sigma subunits in the active site of RNA polymerase. The observation of a small extent of labeling of the beta and alpha subunits, which was prevented by saturating levels of ATP, suggests that these subunits are in close proximity to the catalytic site.

Subunit composition of skeletal muscle transverse tubule calcium channels evaluated with the 1,4-dihydropyridine photoaffinity probe, [3H]azidopine.

“…The finding that more than one component of the channel is photolabelled is not unusual as photoaffinity ligands are known to incorporate into more than one subunit of oligomeric enzymes and hormoneor neurotransmitter receptors (e.g., Hall and Ruoho, 1980;Woody et al, 1984;Yip et al, 1980). This proves that A, B and C decay as independent targets and must be present as such in the membrane.…”

Binding of heat shock proteins to the avian progesterone receptor.

“…They also indicate that the actual ATPbinding component is probably not hsp90, but is likely to be hsp70 or possibly the progesterone receptor proteins themselves, since the binding of progesterone receptor to ATPSepharose has been demonstrated (34). To determine which protein in the receptor complex was binding ATP, we photolabeled the complex with 8-azido[32P]ATP, an effective affinity-labeling agent used in several systems to define ATP-binding proteins (1,2,67). Preliminary studies showed that this ATP analog was bound in a saturable, Mn-dependent manner.…”

5-Azido-2'-deoxyuridine 5'-triphosphate: a photoaffinity-labeling reagent and tool for the enzymatic synthesis of photoactive DNA.

“…Since one of our objectives was to synthesize a photoactive nucleotide analog that would serve as a substrate for DNA synthesis, the allowable modifications were further restricted to those that do not alter the normal anti conformation of the nucleotide. Substituents at the C8 position of purines and the C6 position of pyrimidines usually shift the normal anti conformation toward a syn conformation (16) that renders the nucleotide unacceptable as a substrate for E. coli DNA polymerase I (17) and other template-directed polymerases (7,18,19). Because of this restriction the 8-azidopurine nucleotide analogs are not substrates for template-directed enzymatic synthesis of DNA or RNA.…”