An in vitro system for studying RNA-protein interaction: application to a study of yeast ribosomal protein L1 binding to 5S rRNA.
Previous attempts to study the binding of yeast ribosomal protein L1 with 5S rRNA in vitro have been impeded by the failure to form RNA-protein complexes with purified protein and RNA. To circumvent this difficulty, we have developed an in vitro system that allowed RNP formation. The system involved in vitro expression of the protein L1 from its cloned 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 when 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 those 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 proteins to bind yeast 5S rRNA and concluded that protein L1 missing residues 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 nature of 5S rRNA-protein L1 interaction.
Yeh
L C
LC
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Lee
J C
JC
D011485:Protein Binding; D014176:Protein Biosynthesis; D012331:RNA, Fungal / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D014158:Transcription, Genetic
D012331:RNA, Fungal; D012341:RNA, Ribosomal, 5S; D012269:Ribosomal Proteins