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 (AN-ATP) was found to be reversible in the dark and competitive with ATP, with KI = 2.2 +/- 0.2 microM and KATP = 20 +/- 0.5 microM, but noncompetitive with CTP and GTP, with dissociation constants KCTP = 4.4 +/- 0.4 microM and KGTP = 19.4 +/- 0.7 microM. Under UV irradiation the enzyme was irreversibly labeled by AN-[alpha-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 ATP 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 A as the average distance between them and Kd = 31 +/- 0.1 microM 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 A and Kd = 53 +/- 0.4 microM. These results indicate that the binding of RNA polymerase II to DNA template triggers a conformation change in the enzyme molecule.
Kang
I
I
Department of Chemistry, State University of New York at Buffalo 14214-3094.
Lin
J
J
Wang
J H
JH
D000255:Adenosine Triphosphate / Q000737:chemistry; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000648:ultrastructure; D010777:Photochemistry; D011487:Protein Conformation; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence; D013698:Templates, Genetic
D000345:Affinity Labels; D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D012319:RNA Polymerase II