Isolation and Identification of Membrane Proteins Binding to Transfer RNA

Crude extract of total membrane proteins of yeast was obtained by a method of phase separation in Triton X-114. The membrane extract was applied to the affinity column on which yeast total tRNAs were covalently coupled to hydrazinyl Sepharose 4B. By eluting with increasing concentration gradient of (NH(4))(2)SO(4), eluted proteins were found between concentrations of (NH(4))(2)SO(4) 0.1 mol/L to 0.3 mol/L. By gel mobility-shift assays, it was also observed that there were more than two mobility-shift bands on the gel electropherogram after incubation of sample of (32)P-labeled tRNA with the eluent proteins and the reaction mixtures were analyzed by non-denaturing polyacrylamide gel electrophoresis. These results confirm that these eluted proteins contain two major tRNA specific binding proteins.