FIGURE 3.
In vitro transcription assays with the Rho* mutants. Autoradiograms showing the in vitro Rho-dependent transcription termination assays of the WT and Rho* mutants using the templates having (A) H-19B TR1 and (B) trac terminators. Termination assays were performed both in the presence and absence of NusG. In A, termination zones are marked by double sided arrows. In B, an immobilized DNA template having the trac terminator was used to measure the terminated RNA. The termination zones are indicated by dashed lines. The reactions were fractionated into S (half of the supernatant) and P (half supernatant + pellet) fractions to identify the terminated RNA that appeared as released product. In both the experiments, concentrations of RNAP, Rho, and NusG were 25, 50, and 200 nm, respectively. C, schematic showing the formation of a stalled elongation complex (RB) downstream of the rut sites of the trac terminator using the lac repressor. The elongation complex is immobilized via a 5′-biotin-avidin linkage of the template with magnetic beads. Different components of this stalled complex are indicated. D, autoradiograms showing the time course of RNA release from the EC stalled downstream of the trac terminator sequence as induced by WT and Rho* mutants. RNA release by the WT Rho has been shown both in the presence and absence of NusG. S denotes half of the supernatant and P denotes rest of the sample. RB represents the RNA corresponding to the position of the stalled EC on this template. The 0 min time points are the samples that do not contain Rho. E–G, plots showing the fractions of released RNA against time from the RB, in the presence of WT and Rho* mutants, P235H and R221C both in the presence and absence of NusG as indicated. Average rates of RNA release (k) are indicated inside each of the plots. The experimental data points are fitted as described under ”Materials and Methods.“ Error bars were obtained from three independent measurements.