CsrA Binding to virB 5'UTR RNA

DNA fragments encoding either the entire 5´ UTR of the virB operon or a shortened fragment lacking the first 73 nucleotides, were amplified from X. citri genome using forward primers which include the T7 promoter sequence (see S1 Table). RNA transcripts of the cloned 5´UTR_B7 fragments were produced in vitro from the resulting purified PCR products by using the T7 Transcription Kit (Roche) and labelled by using the RNA 3´ End Biotinylation Kit (Pierce). Recombinant CsrA protein was purified as previously described [27 (link)]. Approximately 70 nM of purified CsrA protein and 6.25 nM Biotin-labeled RNA were mixed with binding buffer (10 mM HEPES (pH 7.3), 20 mM KCl, 1mM MgCl₂, 1 mM DTT, 5% glycerol, 0.1 μg/μL yeast tRNA, 20 U RNasin (Promega)) in a total reaction volume of 20 μL. The binding reactions were incubated at 25°C for 20 min. A 5 μL aliquot of loading buffer (97% glycerol, 0.01% bromophenol blue, 0.01% xylene cyanol) was added to the binding reaction and immediately loaded and resolved by 5% native polyacrylamide gels. The binding assays and detection of RNA products were performed with the LightShift Chemiluminescent RNA EMSA Kit (Thermo Scientific). For the control reactions, 312.5 nM unlabelled (competitor) virB 5´UTR_B7 RNA was added to the binding reactions.