Fig. 1: Reconstitution of vRNAP intermediates.
From: tRNA as an assembly chaperone for a macromolecular transcription-processing complex
a, Schematic representation of the biochemical reconstitution strategy of vRNAP assembly intermediates. Minimal and core vRNAPs were isolated from infected cells by anti-FLAG affinity chromatography; factors D1/D12, E11 and NPH-I were expressed in E. coli and purified as described in the Methods. tRNAGln/Arg was obtained from purified complete vRNAP as described in the Methods. b, Top, silver-stained protein gel of fractions from a sucrose density gradient centrifugation of isolated vRNAP complexes. tRNAGln/Arg from purified vRNAP was visualized by ethidium bromide staining. Fractions 10–12, minimal and core vRNAP sediment; fractions 14–16, complete vRNAP sediments. Bottom, [32P]tRNAGln/Arg was incubated with the indicated vRNAP complexes, separated by gradient centrifugation and analyzed by autoradiography. The experiment was performed in triplicate. c, Gel mobility shift assays of core vRNAP and recombinant factors in the presence of [32P]tRNAGln/Arg. Black dots, added components; white dots, omitted components. The dot size corresponds to the amounts of recombinant factors added. Complex formation was visualized by autoradiography. The experiment was performed four times.
