FIG. 5.
HnRNP U is recruited to the promoter as a part of the PIC and dissociates from the elongating Pol II complex. (A) Anti-hnRNP U coimmunoprecipitates only the IIA form of Pol II in vivo. HeLa whole-cell lysate (lane 1) and the anti-hnRNP U immunoprecipitate (α-HN IP; lane 2) were immunoblotted with anti-CTD antibody 8WG16. (B) The association of hnRNP U with Pol II was monitored in different stages of transcription in vitro as described in Materials and Methods. Transcription reactions were performed with the 5′-biotinylated TK-CAT template and HeLa nuclear extract. Transcription complexes formed from reactions incubated with DNA but without NTPs (lanes 1 and 2), with ATP (lanes 3 and 4), and with all four NTPs (lanes 5 and 6) were immunoprecipitated and immunoblotted as indicated. The hyperphosphorylation of Pol II in the reaction containing NTPs (lanes 5 and 6) was monitored in the presence of [γ32P]ATP.