Volume 132, number 1 FEBS LETTERS September 1981 A QUATERNARY COMPLEX CONSISTING OF TWO MOLECULES OF tRNA AND RIBOSOMAL PROTEINS L2 AND L17 Ene METSPALU, Toivo MAIMETS, Mart USTAV and Richard VILLEMS* Laboratory of Molecular Biology, Tartu University and *Laboratory of Molecular Genetics, Institute of Chemical Physics and Biophysics, 14/16 Kingissepa Street, 202400 Tartu~ Estonian SSR, USSR Received 30 July 1981 1. Introduction Experiments with various affinity and photo-affinity analogues of tRNA have revealed its proximity to a number of ribosomal proteins (reviews [1-3]). More recently, UV-induced in situ crosslinking of tRNA has allowed the establistunent of a group of proteins in the close vicinity of tRNA [4,5]. Direct evidence for the interaction of ribosomal proteins with tRNA was obtained by affinity chromatography of ribosomal proteins on immobilized tRNA [6-10]. The complex of 50 S subunit proteins with tRNA, covalently bound to the epoxy-activated Sepharose is described in [9] and consists of proteins L2, L15, L16, L17, L18, L22, L33 and L34. We have been able to show that this tRNA-pro- tein complex binds another molecule of tRNA and, independently from the latter, a molecule of 5 S RNA [11]. Here we show that two 50 S ribosomal subunit proteins, L2 and L17, bind directly to the immobilized tRNA. Neither of these proteins in sepa- rate dfineric complex with tRNA form a binding site for the second tRNA. However, these two proteins, when taken together, form a complex with immobilized tRNA which possesses a binding site for the second molecule of tRNA. 2. Experimental Escherichia coli tRNA was isolated from MRE600 strain, purified on a Sephadex G-100 column (5 × 90 cm) and its purity was checked by urea- polyacrylamide gel electrophoresis according to [12]. * To whom correspondence should be addressed E. coli 3zp-labelled tRNA was isolated from MRE600 strain, grown on low-phosphate medium containing a/p orthophosphoric acid [ 13]. Hot phenol-SDS- extracted RNAs [ 14] were fractionated by polyacryl- anfide gel electrophoresis [12], eluted from the gel [ 15] and reprecipitated 3 times with alcohol. E. coli 50 S ribosomal subunits were prepared according to [16]. In the isolation and fractionation of 50 S ribo- somal subunit proteins we followed the procedure in [ 17], modified by pre-fractionation of 50 S subunit proteins on a preparative tRNA-Sepharose gel column followed by Sephadex gel fdtration and CM-cellulose chromatography at neutral pH. Details of the proce- dure will be published elsewhere. Individual proteins were identified by two-dimensional polyacrylamide [ 18] and SDS- 15% polyacrylamide gel electrophoresis [191. The binding of the individual proteins, protein mixtures, and 32p4abelled deacylated tRNA to the tRNA-Sepharose gel (53 A260 units/ml) was carried out in 10 mM Tris-HC1 binding buffer (BB) (pH 7.5) containing 10 mM MgClz, 100 mM KC1 and 6 mM 2-mercaptoethanol at 4°C. Proteins were loaded onto 0.5 ml tRNA-Sepharose gel column at ~10 -6 M. The column was washed with 10 column vol. BB, and t[32p]RNA was loaded onto it at 2 × 10 -s M. After that the afffmity column was again washed with BB, until there was no radioactivity in the eluate (about 10 column vol.) and the bound t [32p]RNA with pro- teins were finally eluted with an elution buffer (EB) containing 1 M KC1 and 10 mM EDTA. Radioactivity was measured in an LKB Ultrobeta 1210 scintillation spectrometer with an efficiency of ~40% on 3H-chan- nel for 32P-induced ~erenkov radiation. One-third volume of 30% trichloroacetic acid was added to all affinity column fractions (loading of proteins, loading Published by Elsevier/North-Holland Biomedical Press 00145793181/0000 0000/$02.50 © 1981 Federation of European Biochemical Societies 105