Figure 1.

Transcription elongation pauses in low concentrations of nucleotide precursors. (A) Western blot analysis showed that the concentrated P1.0 fraction contains RNAPII, general transcription factors, Mediator, and P-TEFb, but lacks DSIF and NELF. (B) Schematic representation of pG5MLP, showing the five Gal4-binding sites followed by the adenovirus major late promoter (MLP), and a 380 bp G-free cassette. (C) Transcription assays were carried out using P1.0 and pG5MLP as the template, with 60 μM ATP, 5 μCi of [α-³²P]UTP (800 Ci/mmol) and the indicated concentrations of UTP and CTP. Chase reactions were carried out after 15 min of initiation/elongation, by further incubation for the indicated times with 1.5 mM ATP, CTP and UTP, as shown schematically in the lower diagram. (D) Silver staining of purified recombinant His-DSIF and His-Gal4-VP16 proteins. (E) In vitro transcription assays were carried out using P1.0 and pG5MLP as the template, in the absence or presence of His-DSIF (7.5 ng in lane 2; 30 ng in lane 3; 120 ng in lane 4) and His-Gal4-VP16 (50 ng in lane 5; 100 ng in lane 6; 150 ng in lane 7), as shown schematically in the lower diagram. Numbers to the left indicate the positions of markers (nucleotides). (F) Products in panel E was quantified by a phosphorimager, and the amounts (in arbitrary units) were plotted against the length. The numbers at the top and the bottom indicate the positions of markers (nucleotides). The numbers to the right correspond to the lane numbers in panel E.