FIGURE 4.
NusA-NTD mutants affect in vitro antitermination. Autoradiograms of in vitro transcription termination assays on the templates having H-19B nutR/tR1-trpt′ terminators (A), TR′-T1-T2 terminators (C), and λ tR1 terminator (E) in the presence of WT and NusA mutants with WT H19B-N and λ N. Rho was added for Rho-dependent terminators in A and E. Termination regions are indicated by double arrowed dotted lines next to the transcript bands. At the Rho-independent terminators, specific terminated products are indicated. RO denotes the run-off product. Concentrations of DNA template, RNA polymerase, N, Rho, and NusG were 5, 25, 100, 50, and 200 nm, respectively. Samples were run on an 8% sequencing gels for the templates with Rho-dependent terminators, and that for Rho-independent terminators 6% gel was used. Bar diagrams show the average of read-through efficiencies (% Read-through), those calculated from the in vitro N-induced antitermination assays both at the Rho-dependent (B and F; H-19BtR1-trpt′ and λtR1) and -independent (D) terminators in the presence of the different NusA mutants. Formulae used for the calculations are given under “Experimental Procedures.” The errors were obtained from three measurements.