Binding Affinities for MiDAS RNA Constructs

constants were measured by Christopher W. Leonard (Weeks Lab, UNC) using a dual filter system35 in 50 mM HEPES (pH 7.7), 40 mM potassium acetate (pH 7.7), 0.8 mM MgCl2,0.2

mM DTT, 100 µg/mL BSA and 0.01% (v/v) Triton X-100. Reactions performed without competitor contained 40 pM [32P]-labeled RNA and 0.5-500 nM Gag. Reactions performed in the presence of competitor RNA contained 120 pM [32P]-labeled RNA and a 5000× molar excess of yeast tRNAPhe (Sigma-Aldrich).

Acknowledgements: I would like to thank Julian Bess and Rob Gorelick for providing me

the ex virio dimer extract, for performing SHAPE in virio and for helpful discussions; Sid Datta and Alan Rein for the Gag and NC protein and useful advices about handling the two proteins; and Chris Leonard for painstakingly assessing Gag binding affinities for all the MiDAS truncates and mutants.

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