Figure 4.

Effect of RNA‐IN mutations on Hfq binding.

A. Sequence of the first 50‐nt of RNA‐IN with the RBS and start codon indicated in blue and the sequence of two Hfq‐binding sites (site 1 and site 2) underlined. Nucleotide changes for M5 and M2 mutants are shown.

B. Hfq binding to ³²P‐labeled IN^WT‐160, IN ^M5‐160 and IN ^M2‐160 was measured by EMSA. Binding reactions contained 20 ng μl⁻¹ of total yeast RNA, the indicated concentrations of Hfq (reported per hexamer) and ∼ 1 nM RNA‐IN. Band intensities of the representative gel images shown were quantified and the percent complex formed was plotted against Hfq concentration to calculate apparent dissociation constants (K_D). Hfq‐RNA‐IN interactions for IN:Hfq‐1 and IN:Hfq‐2 are described by K _D1 and K _D2 respectively.