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-naphthalenesulf onyl)-ATP (AN-ATP) was found to be reversible in the dark and competit ive with ATP, with K-I = 2.2 +/- 0.2 mu M and K-ATP = 20 +/- 0.5 mu M, but noncompetitive with CTP and GTP, with dissociation constants K-CT P = 4.4 +/- 0.4 mu M and K-GTP = 19.4 +/- 0.7 mu M. Under UV irradiati on the enzyme was irreversibly labeled by AN-[alpha-P-32]ATP. A linear plot of the relative specific activities of labeled enzyme samples af ter gel filtration vs the number of labels per enzyme molecule shows t hat each covalent label completely inactivates an enzyme molecule. The refore the labeling has maximal specificity at an essential specific A TP site which is not the substrate-binding site for the polymerizatio n reaction. The fluorescent AN-ATP-labeled enzyme, with absorbance max imum at 310 nm and emission maximum at 370 nm, can still bind noncoval ently a second ligand, 2'-O-dansyl-ATP, with absorbance maximum at 365 nm and emission maximum at 560 nm, at the substrate-binding site. Mea surement of energy transfer between the two fluorescent labels gave R = 23.8 +/- 0.4 Angstrom as the average distance between them and K-d = 31 +/- 0.1 mu M for the bound 2'-O-dansyl-ATP in the absence of DNA t emplate. The addition of either thymus DNA or poly [d(A-T)] to the sys tem changed these values to R = 25.6 +/- 0.4 Angstrom and K-d = 53 +/- 0.4 mu M. These results indicate that the binding of RNA polymerase I I to DNA template triggers a conformation change in the enzyme molecul e.