La-Module Binding Kinetics with RNA

RNA oligonucleotides were 5ʹ-end labelled with [γ-³²P]-ATP (Perkin Elmer) using T4-polynucleotide kinase (New England Biolabs) and gel purified. Capped RPS6 20-mer was prepared as previously described[25 (link)]. La-Module 5X stocks were prepared in dilution buffer (50 mM Tris-HCl pH, 7.5, 100 mM NaCl, 25% glycerol, 4 mM DTT). 10 μL binding reactions contained final concentrations of 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 7.5% v/v glycerol, 1mM DTT, 1 μM BSA (Thermo Fisher Scientific), ≤ 2 nM radiolabeled RNA, and either: 10 U/mL poly(dI-dC) (Sigma-Aldrich), 5 μM Yeast tRNA (Ambion) or 3 μM salmon sperm DNA (Thermo Fisher Scientific). La-Module was titrated at 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 μM. Reactions were incubated on ice for 30 min then run on 7% polyacrylamide (29:1) native 0.5X TBE gels at 125 V for 45 min at 4°C. Exposed phosphor screens (GE Healthcare Lifesciences) were imaged on a Typhoon FLA plate reader (GE Healthcare Lifesciences) and quantitated using Imagequant TL (GE Healthcare Lifesciences). Dissociation constants were determined by plotting (KaleidaGraph) the fraction of shifted RNA versus the concentration of protein after band intensities were corrected for background (ImageQuant)TL.