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. 2024 Mar 20;628(8006):186–194. doi: 10.1038/s41586-024-07206-5

Fig. 5. Compensatory mutations in nusG increase the fitness of βS450L Mtb.

Fig. 5

a, Experimental design to quantify the competitive fitness of nusG, rpoB and rpoC mutants in RifS and βS450L Mtb. (1) Generation of barcode (BC) library; asterisk indicates premature stop codon. (2) ssDNA recombineering to generate mutants of interest. (3) Pooled, competitive growth experiment. Oligo, oligonucleotide. b,c, Competitive index (mean ± s.d. from 3 biological replicates) of each mutant relative to the RifS base strain (no mutations in nusG, rpoB or rpoC). The competitive index of each mutant was normalized to its respective base strain, RifS or βS450L, by subtracting the competitive index of the base strain. nusG-T200A is a control mutation not associated with RifR in clinical isolates and is thus not expected to restore fitness to βS450L Mtb. For reference, the fitness of βS450L relative to RifS in this experiment is 0.96. Paired two-sided t-test. **P ≤ 0.01, ***P ≤ 0.001. d, Model explaining the mechanism of reduced fitness of βS450L Mtb and its compensation. The βS450L RNAP elongates more slowly and is thus more likely to swivel and enter the paused and termination states. This effect is exacerbated by the pro-pausing wild-type NusG, as illustrated by an increase in the activation barrier of elongation. These interactions decrease βS450L Mtb fitness. Note that the competitive index values shown here are not base-subtracted as they are in b,c. Compensatory mutations at the NusG–β-protrusion or NusG–NT-DNA interface reduce the ability of NusG to stabilize the swivelled state, as illustrated by a relative decrease in the activation barrier of elongation. These mutations restore βS450L RNAP pausing and termination levels closer to wild-type RNAP and increase βS450L Mtb fitness.