Fig. 1. PutL-mediated suppression of a U-rich pause.
A. A diagram of folded putL RNA and the U-rich pause. The HK022 PL-proximal transcript, which includes the two stem-loops that comprise putL RNA and the downstream U-rich pause, is shown. The RNA segment within an EC paused at position G93 is indicated by nucleotides shown with a gray background. The sequence of the antitermination-defective putL mutation used in most experiments is shown [mutant G of (King et al., 1996)]. B. The effect of antitermination-defective mutations in putL and RNAP on pause intensity. Lanes 1–4: The template encoding WT or mutant putL was transcribed by WT or mutant RNAP for 3 min in the presence of 100 µM ATP, UTP, GTP and 25 µM CTP. Lane 5: An EC roadblocked by Lac repressor at the pause site was obtained as described in Methods. C. Mapping the pause sites by RNAP walking. An EC roadblocked at the pause site (lane 1) was incubated in the presence of the indicated NTPs after the roadblock was removed with IPTG (lanes 2–6). D. Backtracked and pretranslocated states of an EC stalled at position G93. The grey oval represents RNAP and the grey dot, the active center.