FIGURE 9.

Functional interaction sites of NusG on the Rho hexamer. A, the scheme describing the CTD toxicity assay. The CTD of NusG has more affinity for the Rho than the full-length NusG, and therefore, its overexpression titrates out the latter from the elongation complex leading to conditional toxicity of the cells. Schematics show the representation of the toxicity of the overexpressed NusG-CTD in the presence of WT Rho. B, amino acid sequences of the region of Rho that cross-links to the NusG-CTD (9). The positions of the point mutations in this region are defective in Rho binding in vitro. C, MG1655Δrho strains (RS1305) expressing WT and the indicated Rho mutants from the pCL1920 plasmids were transformed with arabinose-inducible pHYD3011 (a modified pBAD plasmid) plasmid expressing the NusG-CTD fragment. Different dilutions of the overnight culture of the strains were spotted on LB plates either in the absence (0%) or presence of 0.2% arabinose to check the growth defects by the overexpression of the NusG-CTD fragment. Dilutions are indicated on the left side of the figures, which indicate the platting efficiencies. D, growth characteristics of MC4100ΔracΔrho strains having either WT or G146D or L158Q nusG mutants in the chromosome in the presence of different Rho derivatives, WT, V203L, M205A, K224C, and V228N expressed from low-copy pCL1920 plasmids. After transforming with the Rho plasmids, the chromosomal rho was removed by P1 transduction and the transductants were re-streaked on the LB plates to check the synthetic lethality.