FIG. 4.

Transcriptional activation by selected mutant NtrC proteins in vitro. Formation of open complexes by ς⁵⁴ holoenzyme was assessed by a single-round transcription assay as described in Materials and Methods. NtrC proteins were present at the concentrations indicated and were phosphorylated with carbamyl phosphate (10 mM). Effects of single amino acid substitutions were assessed by comparison to NtrC^WT (open circles) in each panel. (A) V177A and I200V. (B) A220V. (C) Q230R and M244V. (D) Y262C and G291R. Proteins were chosen to illustrate their hyperactivity at low concentrations and the widespread locations of their amino acid substitutions within the central domain of NtrC: V177A between conserved regions 1 and 2 (C1-C2), I200V (C2), A220V (C3), Q230R (C3-C4), M244V (C4-C5), Y262C (C5), and G291R (C6). Data for NtrC^WT is included in each plot to illustrate the variability between experiments.