RNA emerging from the active site of RNA polymerase II interacts with the Rpb7 subunit

Abstract

Structural studies of RNA polymerase II have suggested two possible exit paths for the nascent RNA: groove 1, which points toward the subcomplex of subunits Rpb4 and Rpb7, and groove 2, which points toward Rpb8. These alternatives could not be distinguished previously because less than 10 nucleotides (nt) of transcript were resolved in the structures. We have approached this question by UV cross-linking nascent RNA to components of the transcription complex through uridine analogs located within the first six nucleotides of the RNA. We find that the emerging transcript cross-links to the Rpb7 subunit of RNA polymerase II in various complexes containing 26- to 32-nt transcripts. This interaction is greatly reduced in complexes with 41- or 43-nt RNAs and absent when the transcript is 125 nt. Our results are consistent with groove 1 being the exit path for nascent RNA.

Functional Interactions of the RNA Polymerase II-interacting Proteins Gdown1 and TFIIF

“…TFIIH was purified from HeLa nuclear extract by chromatography on P11 phosphocellulose, DE52, and Mono Q as described (15). RNA polymerase II was purified from the initial chromatin pellets obtained during preparation of HeLa nuclear extract by chromatography on DE52, heparin Sepharose and Mono Q using the method of Maldonado et al (22) with modifications as described (21). Recombinant His-tagged Gdown1 was expressed in E. coli and purified on nickel-nitrilotriacetic acid resin before purification on a Mono Q column (3).…”

“…Proteins and Factors-Recombinant TATA box binding protein (TBP), TFIIB, and TFIIE were prepared as described (20,21). Recombinant human TFIIF subunits, including the truncated 1-227 large subunit, were expressed, purified, and assembled as described (15).…”

Efficient and Rapid Nucleosome Traversal by RNA Polymerase II Depends on a Combination of Transcript Elongation Factors

“…TFIIH activity eluted from Mono Q between 0.25 and 0.35 M KCl. Human pol II was purified from HeLa cell nuclear pellets by the protocol of Maldonado et al (37) with the modifications described by Ú jvári and Luse (33). Human TFIIS was a recombinant GST fusion protein purified by binding and elution from glutathione-agarose.…”

“…Proteins and Factors-The human general transcription factors TATA-binding protein, TFIIB, and TFIIE were recombinant proteins prepared as described (33,34). Recombinant human TFIIF subunits were expressed, purified, and assembled as described by Wang et al (35,36) with the addition of a size-exclusion chromatography step at the end of the purification.…”

Inactivated RNA Polymerase II Open Complexes Can Be Reactivated with TFIIE

“…TFIIH was isolated from HeLa nuclear extract as reported (21), and RNA polymerase II was purified from HeLa nuclear pellets as described (23,24). TFIIF was phosphorylated in vitro with casein kinase 2 and subsequently purified by the method of Újvári et al (25) with minor modifications as described (22).…”