Quantitative CsrA-RNA Binding Assay

Binding of CsrA to RNAs was determined by EMSA with recombinant CsrA-His₆^{17 (link)} and RNA synthesized in vitro with MEGAshortscript Kit (Ambion) or the Ampliscribe T7 flash Transcription Kit (EpiCentre). The template DNA for in vitro transcription of sRNAs was generated by PCR from MG1655 genomic DNA, using the primers listed in Supplementary Data 10. In vitro-transcribed sRNAs were gel-purified, treated with Antarctic phosphatase (NEB), and radiolabeled at the 5′ end using [γ-³²P] ATP and T4 polynucleotide kinase. Binding reactions contained 0.1–0.5 nM RNA, 10 mM MgCl₂, 100 mM KCl, 32.5 ng total yeast RNA, 20 mM DTT, 7.5% glycerol, 4 U SUPERasin (Ambion), and various concentrations of CsrA (0–400 nM), and incubated at 37 °C for 30 min. Reactions mixtures were separated on native polyacrylamide gels using 1× TBE as the electrophoresis buffer and labeled RNA was analyzed using a PMI phosphorimager (Bio-Rad) and Quantity One software (Bio-Rad), as described previously^{17 (link)}. Apparent equilibrium binding constants (K_d) presented in the figures are averages of two independent experiments.