A complex between immobilized tRNA and Escherichiu coli 50-S ribosomal subunit proteins provides binding sites for several other components of the protein biosynthesis system. We found that this complex binds 5-S RNA, tRNA and 30-S ribosomal subunits. The binding of 5-S RNA and tRNA takes place at equilibrium; in 10 mM Tris/HCl buffer, pH 7.5, containing 10 mM MgC12 and 100 mM KCI at 4 "C it is characterized by the dissociation constants 1.7 x M and 4 x lo-' M respectively. We show that the perfornicd complex between tRNA and 5 0 3 ribosomal subunit protein can bind these two RNAs simultaneously by forining a subribosoinal domain consisting of two molecules of tRNA, 5-S R N A and the ribOSomd1 proteins L2. L15, L16, L17, L18, L22, L33 and L34. The preformed t R N A . protein complex interacts also with the 30-S ribosomal subunit, suggesting that it is located at the ribosomal interface.A number of recent reviews deal with various aspects of the interaction between tRNA and the ribosomes [I -51. In spite of its comparatively long history, the mechanism of the tRNAjribosome interaction is still largely an open question. What we know about tRNA interaction with the components of the ribosome (ribosomal proteins and rRNA) comes from results obtained by indirect methods and can be, therefore, considered only as the first approximation when we ask a question about the direct contacts between tRNA and ribosomes. Four laboratories have published results showing interactions between ribosomal proteins and immobilized tRNA [6-131. These results demonstrate the possibility, but only the possibility, of direct interactions between certain ribosomal proteins and tRNA.We argued earlier [8-111 that the proteins forming a complex with the immobilized tRNA are those located at the ribosomal decoding site or in the vicinity of the peptidyl transferase center. In a more recent study [12] we showed that the complex of 5-S RNA with 50-S ribosomal subunit proteins binds one molecule of tRNA. Here we extend this observation and describe subribosomal complex containing ribosomal proteins and two molecules of tRNA as well as 5-S RNA. MATERIALS A N D METHODS Isolation OfrRNA, 5-S R N A 50-S Ribosomal Subunit Proteins und 30-S SubunitsAll these components have been isolated from Escherichiu coli MRE600 strain according to published procedures [8 -10, 12,141. The purity of tRNA and 5-S RNA was controlled byAbbreviations. TP50, total protein fraction of E. co/i 5 0 4 ribosomal subunit; bisoxyrane, 1,4-bis(2,3-epoxypropoxy)butane.Dcjinition. 1 AZ60 unit, the quantity of RNA in 1 ml solution which has an absorbance of 1 at 260 nm, when measured in a I-cm pathlength cell. urea/polyacrylamide gel electrophoresi\ according to Maxam and Gilbert [I 51. Ribosomes and their subunits were isolated as described in [I61 and 50-S subunit proteins were extracted by acetic acid [17]. We also isolated tKNA from baker's yeast by the phenol/sodium dodecyl sulphate method followed by further purification on DEAF.-Sephadex A-50 chromatography and Sephadex G-1...