Binding of Ribosomal Proteins to RNA Covalently Coupled to Agarose

An affinity chromatographic procedure, using ribosomal or tRNA covalently coupled to agarose through an adipic acid dihydrazide spacer, has been developed to isolate and identify the Escherichia coli ribosomal proteins which specifically bind to these RNA molecules. Transfer RNA, 5-S, 16-S and 23-S ribosomal RNA were readily immobilized on agarose-dihydrazide; the maximum amount coupled decreasing with increasing molecular size. Specific binding of ribosomal proteins to RNA was observed in buffer containing 0.3 M KCl and 0.02 M MgCl2. Bound proteins were eluted with a high-salt, 2 M KCl, buffer containing 0.005 M EDTA. Two ribosomal proteins, identified as L18 and L25 by two-dimensional gel electrophoresis, bound tightly to E. coli 5-S RNA; small amounts of a third protein, L5, also were bound. These proteins did not bind to immobilized tRNA nor did 30-S ribosomal proteins bind to 5-S RNA. Denatured 5-S RNA bound less L18 and L25 than the native species. Several 30-S and 50-S ribosomal proteins bind to immobilized tRNA. The major 50-S subunit proteins tightly bound to tRNA were identified as L3, L4, L5, L7 and L8/L9. Smaller amounts of proteins L1, L2, L11, L16 and L21 were also observed. Proteins L2 and L16 were retarded by agarose-bound tRNA. Protein S3 was the major 30-S subunit protein bound to tRNA. Lesser quantities of proteins S6, S9, S13 and S18 also interacted with tRNA.