Affinity labeling and measurement of DNA-induced conformation change in RNA polymerase II

The inhibition of RNA polymerase II by 8-azido-2′-O-(1-naphthalenesulfonyl)-ATP (ANATP) was found to be reversible in the dark and competitive with ATP, with K₁ = 2.2 ± 0.2 μM and K_ATP = 20 ± 0.5 μM, but noncompetitive with CTP and GTP, with dissociation constants K_CTP = 4.4 ± 0.4 μM and K_GTP = 19.4 ± 0.7 μM. Under UV irradiation the enzyme was irreversibly labeled by AN-[α-³²P] ATP. A linear plot of the relative specific activities of labeled enzyme samples after gel filtration vs the number of labels per enzyme molecule shows that each covalent label completely inactivates an enzyme molecule. Therefore the labeling has maximal specificity at an essential specific A TP site which is not the substrate binding site for the polymerization reaction. The fluorescent AN-ATP-labeled enzyme, with absorbance maximum at 310 nm and emission maximum at 370 nm, can still bind noncovalently a second ligand, 2′-O-dansyl-ATP, with absorbance maximum at 365 nm and emission maximum at 560 nm, at the substrate binding site. Measurement of energy transfer between the two fluorescent labels gave R = 23.8 ± 0.4 Å as the average distance between them and K_d = 31 ± 0.1 μM for the bound 2′-O-dansyl-ATP in the absence of DNA template. The addition of either thymus DNA or poly [d(A-T)] to the system changed these values to R = 25.6 ± 0.4 Å and K_d = 53 ± 0.4 μM. These results indicate that the binding of RNA polymerase II to DNA template triggers a conformation change in the enzyme molecule.