Fig. S3.

Mutation in rut disrupts Rho binding to the mgtA leader RNA, sequences important for RARE activity in the mgtCBR leader. (A) Rho binding to the mgtA leader RNA variants (3′ segment 243 nt long) or control RNA. The C145G mgtA variant promotes an RNA conformation favorable for Rho binding (8). The R1 mgtA variant has substitutions in rut (8). A ³²P-labeled fragment of mgtA leader RNA variants or control RNA corresponding to a 3′ region of gfp ORF was incubated with increasing concentrations of Rho, and protein-bound RNA fractions were separated by filtering. The amount of filter-bound ³²P-RNA was plotted against the concentration of Rho, and an apparent K_d was determined by fitting data to a hyperbolic equation (see Materials and Methods for details). Each data point is an average of at least three independent experiments. (B) Relative values of fluorescence produced by wild-type Salmonella harboring a plasmid that contains a transcriptional fusion between the wild-type mgtCBR promoter and an mgtCBR leader variant lacking nucleotides 80–89 so that RARE is locked single stranded (or having single nucleotide derivatives in RARE), to a promotorless gfp gene. Bacteria were grown as described in the legend to Fig. 4B. Fluorescence produced by wild-type Salmonella strains harboring the RNA variant lacking nucleotides 80–89 was set as 100%. Colors reflect the severity of the effect caused by the mutations (see key to the Right of the table). Data correspond to the average from at least three independent experiments.