Proteins of Small Subunits of Rat Liver Ribosomes that Interact with Poly(U)

II. Cross-links between Poly(U) and Ribosomal Proteins in 40 S Subunits Induced by UV Irradiation

Abstract

(1) When rat liver 40 S ribosomal proteins in 6 M urea were mixed with poly(U) at an appro-priate ratio, a precipitate was formed which was also insoluble in the sample solution for two-dimensional acrylamide gel electrophoresis. Analyses by two-dimensional acrylamide gel electrophoresis showed that S7 and SIO proteins (according to our numbering system) had disappeared selectively from the fraction soluble in 6 M urea. These two proteins were present in the fraction insoluble in 6 M urea, and became soluble in the sample solution after treating it with RNase. The results suggest that S7 and SIO proteins have strong affinities for poly(U). When rat liver 40 S subunits were incubated with poly(U), similar results were obtained.
(2) After incubation of 40 S subunits with [³H]poly(U) and then with unlabeled poly(U), UV irradiation cross-linked poly(U) to the protein moiety of the 40 S subunit. When the protein fraction insoluble in the sample solution for two-dimensional electrophoresis was prepared from 40 S subunits cross-linked to poly(U) and then subjected to two-dimensional acrylamide gel electrophoresis after RNase treatment, S7 and S10 proteins were detected on the gel. In addition to the S7 protein spot, a triangular area spreading from the spot to the origin contained radioactivity. The results suggest that poly(U) is cross-linked to S7 protein and oligo(U) fragments bound to S7 protein affect its electrophoretic mobility.
(3) Ribosomal proteins were prepared from 40S subunits cross-linked to carrier-free [³H]poly(U) and analyzed by three-dimensional acrylamide gel electrophoresis (Terao, K. & Ogata, K. (1975) Biochim. Biophys. Acta 402, 214-229) after RNase treatment. It was found that S7, S6, and S15 proteins are cross-linked to poly(U). From the results of the present and preceding experiments it is concluded that S7 is the poly(U)-binding protein. The possibility that other proteins in 40 S ribosomal subunits interact with poly(U) is discussed.