Journal: The Journal of Biological Chemistry

Article Title: Nascent RNA Cleavage by Arrested RNA Polymerase II Does Not Require Upstream Translocation of the Elongation Complex on DNA *

doi:

Figure Lengend Snippet: A, time course of SII-dependent elongation from site Ia in the presence of all four nucleotides. Washed complexes (lane Ia) were split into 2 aliquots. One received bovine brain SII and 7 mM MgCl2 and was incubated at 28 °C for 1.5 or 15 min to generate the first (*) and second (**) cleavage intermediates, respectively. The second aliquot of washed complexes received bovine brain SII, MgCl2, and 800 μM each of all four NTPs. Portions of this reaction were stopped after the indicated times at 28 °C and analyzed by electrophoresis with the first and second cleavage intermediates. RO, runoff RNA. B, RNA elongation by an SII-independent elongation complex in the presence of SII. RNA in washed complexes was extended for 10 min at 28 °C to positions G218/G220 (indicated by dash at left, lane 0) in the presence of UTP, CTP, and GTP (800 μM each), bovine brain SII, and 7 mM MgCl2. The reaction was chilled to 4 °C, ATP (800 μM) was added, and samples were stopped at the indicated times after incubation at 28 °C. One sample (sar) was adjusted to 0.25% in Sarkosyl and another (α) to 1 μg/ml in α-amanitin before the addition of ATP and incubation at 28 °C. Arrowheads indicate the position of marker RNAs of 260, 380, 420, and 540 nucleotides (bottom to top). C, RNA elongation by a second SII-independent elongation complex in the presence of SII. Elongation complexes were assembled at site Ia (Ia) and moved to positions G218/G220 (dash to left of figure) as described in the legend to B. These complexes were washed free of nucleotides by centrifugation and resuspension and moved to position C230 (U) after an 8-min incubation at 28 °C in the presence of bovine brain SII, 7 mM MgCl2, and 800 μM each of ATP, GTP, and CTP. The reaction was incubated at 28 °C with UTP (800 μM) for the indicated times. One sample (sar) was made 0.25% in Sarkosyl before the addition of UTP and incubation at 28 °C.

Article Snippet: Fast protein liquid chromatography-purified NTPs, 4 ddNTPs, and 3′- O -methyl GTP were purchased from Pharmacia LKB Biotechnology Inc., 3′-dUTP was purchased from Boehringer Mannheim.

Techniques: Incubation, Electrophoresis, Marker, Centrifugation