AN IN-VITRO SYSTEM FOR STUDYING RNA-PROTEIN INTERACTION - APPLICATIONTO A STUDY OF YEAST RIBOSOMAL-PROTEIN L1 BINDING TO 5S RIBOSOMAL-RNA

Previous attempts to study the binding of yeast ribosomal protein L1 w ith 5S rRNA in vitro have been impeded by the failure to form RNA-prot ein complexes with purified protein and RNA. To circumvent this diffic ulty, we have developed an in vitro system that allowed RNP formation. The system involved in vitro expression of the protein L1 from its cl oned gene in the presence of exogenous yeast 5S rRNA. A protein of the expected size (34 kDa) was synthesized by in vitro transcription and translation. A specific 5S rRNA-protein L1 complex (RNP) was formed wh en the rRNA molecule was present during protein L1 synthesis. However, the full-length protein L1 failed to bind 5S rRNA. The extent of RNP formation was proportional to the concentration of the exogenous yeast 5S rRNA in the reaction. The RNP displayed properties identical to th ose isolated from mature 60S ribosome subunits. Addition of yeast 5.8S rRNA did not result in the formation of a specific RNP. Using this in vitro system, we examined the ability of several deletion mutant prot eins to bind yeast 5S rRNA and concluded that protein L1 missing resid ues 261 to 295 from the C-terminus could not bind yeast 5S rRNA. This in vitro system should be useful for future studies on the molecular n ature of 5S rRNA-protein L1 interaction.