Figure 2.

Assaying for intrinsic termination at the λ tR2 terminator on both ss- and dsDNA templates. (A) Stalled C₄₇ complexes (lanes 1 and 5) in TGK-B₄₀M₁ were chased with 500 μM ATP, 500 μM GTP, 500 μM CTP, and 500 μM UTP for 10 min at 42°C in the presence of rifampicin at 20 μg/ml (lanes 2–4 and 6–8). Lanes 2 and 6 are aliquots of the chase reactions before the pellet was separated from the supernatant fluid, lanes 3 and 7 are the RNAs associated with the Dynabeads (P), and lanes 4 and 8 are the RNAs released into the supernatant fluid (S). The λ tR2 terminator is at positions +104 to +105 relative to the transcription start site, whereas the end of the DNA template or run-off occurs at position +177. Lanes: 1–4, no Exo III; 5–8, digested with Exo III (2,000 units/ml) for 5 min at 30°C. Quantitation of the total counts within a lane between the C₄₇ complex and the run-off transcript revealed that equivalent counts were present in lanes 1 and 2 and in lanes 5 and 6. One interesting observation we made was that the majority of the run-off transcripts, particularly in the case of the double-stranded chase reaction, remained associated with the pellet (compare lanes 3 and 4). A plausible explanation for this is that the RNAs associated with the pellet are part of ternary complexes that have arrested proximal to the end of the linear DNA template. This type of phenomenon has also been reported for mammalian RNA polymerase II (28). (B) Exo III-digested DNA was analyzed on a 6% denaturing PAGE gel and detected by chemiluminescence. The nontemplate strand was end-labeled with on the 5′ end with a biotin moiety. Lanes: 1, no Exo III; 2, Exo III at 2,000 units/ml for 5 min at 30°C. The initial DNA template, 315 nucleotides long was reduced to approximately 204 nucleotides after Exo III digestion.