Figure 4: Mutations in β-SI2 have distinct phenotypes that include hyper-attenuation at the trp locus.

A) β-ΔSI2 has mild sensitivity to ethanol. Left: sublethal doses of ethanol hampered growth of the parental strain starting during the transition to stationary phase. Right: β-ΔSI2 has a more pronounced response to ethanol that begins at a lower concentration of ethanol.

B) β-ΔSI2 has mild sensitivity to ethanol, trimethoprim, and hydroxyurea. The normalized area under the curve (AUC) of growth curves as shown in (A) was calculated by integrating OD₆₀₀ over time and normalizing by the AUC of the same strain without added drug. For ethanol (top), trimethoprim (middle), and hydroxyurea (bottom), growth of β-ΔSI2 was affected at lower (sub-inhibitory) concentrations, but the minimum inhibitory concentration remained the same. Error bars represent 95% confidence intervals. Drug concentrations for which the difference in relative AUC between β-ΔSI2 and its parental control is statistically significant are marked with asterisks (p<0.05).

C) Mutant-mutant correlations show a statistically significant association with distance between the residues in the RNA polymerase structure. Mutant-mutant correlations were calculated using Pearson’s r from the chemical-genetic dataset. Residue-residue distance was calculated based on the linear distance between alpha carbons of residues with mutations in the dataset as determined from the 3-dimensional structure. The PDB structure 4JKR was used for distance calculations. A three-mutant clique comprised of β-I966S, β′-N458A, and β′-P1022L was an exception to this rule, with high mutant-mutant correlations despite containing the largest inter-residue distance in the library (β-I966S to β′-P1022L).

D) Correlations among β-I966S, β′-N458A, and β′-P1022L were predictive of a shared hyper-attenuation phenotype that was originally identified for β′-P1022L (Weilbaecher et al., 1994). In a ΔtrpR background, expression of the trp locus is mainly controlled by attenuation. Hyper-attenuation reduces trp expression and makes cells resistant to a toxic analogue of a tryptophan biosynthesis intermediate, 5-methyl anthranilic acid (5-MAA) at 100 μg/mL.