Identification of a nucleoside triphosphate binding site on calf thymus RNA polymerase II

Abstract

A nucleoside triphosphate binding site on calf thymus RNA polymerase II was identified by using photoaffinity analogues of adenosine 5'-triphosphate and guanosine 5'-triphosphate. Both radiolabeled 8-azidoadenosine 5'-triphosphate (8-N3ATP) and radiolabeled 8-azidoguanosine 5'-triphosphate (8-N3GTP) bound to a single polypeptide of this enzyme. This polypeptide has a molecular mass of 37 kilodaltons and an isoelectric point of 5.4. Ultraviolet (UV) irradiation was necessary for photolabeling to occur. In addition, no labeling occurred when the probe was prephotolyzed or when the enzyme was inactivated. Furthermore, photolabeling of the enzyme could be decreased by preincubation with natural substrates. To provide evidence that the radiolabeled polypeptide forms a part of the domain of the nucleoside triphosphate binding site, experiments were performed using unlabeled 8-N3ATP. Although this unlabeled analogue was not a substrate for RNA polymerase II, it photoinactivated the enzyme in the presence of UV irradiation, and it inhibited transcription elongation by the enzyme in a competitive manner in the absence of UV irradiation. As in the case with photolabeling, photoinactivation by 8-N3ATP could be decreased by natural substrates; in both cases, purine ribonucleoside triphosphates were more efficient than pyrimidine nucleoside triphosphates. Furthermore, photoinactivation was saturable at about the same concentration as the inhibition constant for 8-N3ATP. Collectively, these results provide evidence that the radiolabeled polypeptide in calf thymus RNA polymerase II is an essential component for activity and suggest that this polypeptide may be part of this enzyme's purine ribonucleoside triphosphate binding site.

Characterization of RNA polymerase type II from human term placenta

“…The apparent KM for UTP and ATP on denatured calf thymus DNA template were determined for placental RNA polymerase type 11. Upon comparison of these results with those obtained with calf thymus RNA polymerase type 11, the apparent KM for the pyrimidine nucleotide UTP is again lower than the apparent KM for the purine nucleotide ATP (Freund and McGuire, 1986). The turnover numbers are comparable for UTP, but lower for placental RNA polymerase type I1 when the AMP incorporation was followed.…”

“…The crude homogenate of 350 gm placenta contained 28 gm of protein with at least 1,177 units of RNA polymerase type I1 activity (see Table l), that is, with a specific activity of 0.042 units of RNA polymerase type I1 activity per mg protein, which is comparable to the specific activity of 0.045 units of RNA polymerase type II activity per mg protein in the crude homogenate of the calf thymus (Freund and McGuire, 1986). After purification of the placental polymerase type 11, the specific activity was 169 units per mg protein, indicating a purification factor of approximately 4,000.…”

“…Recent experiments on the nucleoside triphosphate binding site of prokaryotic RNA polymerase (Woody et al, 1984) and of calf thymus RNA polymerase type I1 (Freund and McGuire, 1986), using radiolabeled azido ribonucleoside triphosphates as photoprobes, have demonstrated that unlike the prokaryotic enzyme where several polypeptides are labeled, only polypeptide E of the calf thymus is labeled with these probes. It was of interest, therefore, to compare the labeling pattern of placental RNA polymerase type I1 with that of the calf thymus enzyme.…”

“…All operations were performed under a red safety light as detailed previously (Freund and McGuire, 1986). The indicated amounts of [ Y -~~P ] -~-N~A T P / G T P were added to 1.5 ml microcentrifuge tubes, and the solvent methanol removed by a stream of nitrogen.…”

Functional Organization of Two Large Subunits of the Fission Yeast Schizosaccharomyces pombe RNA Polymerase II

“…In this study, we tried to locate the active center for RNA polymerization using the method of affinity labeling with photoreactive nucleotide analogues. This technique has been used to identify the active sites of Escherichia coli RNA polymerase (10 -13), calf thymus RNA polymerase II (14), Saccharomyces cerevisiae RNA polymerase I(A), II(B), and III(C) (15)(16)(17)(18), HeLa RNA polymerase II (19), wheat germ RNA polymerase II (18,20), and influenza virus RNA polymerase (21). Depending on experimental systems such as the type of enzymes and templates, the structure of affinity reagents and the length of nascent RNA chains, the cross-linking has been observed at different portions of the respective RNA polymerases, but always on one or both of the two large subunits, i.e.…”