Fig 4. Gdown1 does not functionally associate with Pol II in open complex or with initiating Pol II.

(A) PICs on bead bound AdML 31g(M) templates were converted to open complexes by incubation with ATP for 5 min, along with 240 fmole Gdown1 (lanes 3 and 4 only) as described in Materials and Methods. After the supernates were removed and replaced with buffer M5, reactions were supplemented with TFIIE and EECs were generated by pulse labeling with ATP, UTP and [α-³²P]CTP as described in Materials and Methods. Reactions were either stopped at this point (P lanes) or supplemented with TFIIF and chased (C lanes) for 60 sec with 200 μM NTPs as described in Materials and Methods. Lengths of markers are shown on the left edge of the gel and TFIIF-dependent and TFIIF-independent transcript lengths are noted on the right. A schematic of the assay is shown to the left of the gel. The gel shown is representative of three replicates that were performed. (B) For lanes 4–7, complexes undergoing abortive initiation were generated on bead bound AdML 31g(M) templates with CpA and CTP as described in Materials and Methods. Reactions in lanes 6 and 7 contained 240 fmol of Gdown1. After 5 minutes the supernatant was removed and replaced with MEMDM40. EECs were then generated with ATP, UTP and [α-³²P]CTP as described in Materials and Methods. Reactions were either stopped at this point (P lanes) or supplemented with TFIIF and chased (C lanes) for 60 sec with 200 μM NTPs. Control reactions shown in lanes 1–3 were performed as described in Fig 3 (EEC lanes). The lane 1 reaction was stopped after the pulse (P) step; for the two chase (C) reactions, Gdown1 was incubated for 5 min with the 30-mer complexes only for lane 3. Lengths of markers are shown on the left edge of the gel, and lengths of TFIIF-dependent and TFIIF-independent transcripts are noted on the right. A schematic of the assay in lanes 4–7 is shown to the left of the gel. The gel shown is representative of three replicates that were performed.