Ene Metspalu

The properties of the complex between ribosomal protein L2 and tRNA

Escherichia coli ribosomal protein L2 interacts with fMet‐tRNAMet and NacPhe‐tRNAPhe in solution, protecting their 3‐ends from enzymatic degradation. At the same time L2 enhances the rate of spontaneous hydrolysis of the ester bonds between terminal riboses and amino acyl moieties of these two peptidyl‐tRNA analogues. L2 has, however, only a slight effect on the rate of spontaneous deacylation of aminoacyltRNAs. We suggest that the role of L2 is in the fixation of the aminoacyl stem of tRNA to the ribosome at its P‐site, and speculate that this protein is directly involved in the peptidyl transferase (PT) reaction. Peptidyl transferase Protein L2 tRNA‐protein complex

A QUATERNARY COMPLEX CONSISTING OF TWO MOLECULES OF tRNA AND RIBOSOMAL PROTEINS L2 AND L17

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/

5S RNA‐Protein complex is involved in ribosomal subunit association

02.50

High impact of genetic drift on the isolated population of Rab Island – evidence from mitochondrial DNA diversity

The immobilized tRNA‐50 S ribosomal subunit protein (TP50) complex binds the smaller ribosomal subunit. We constructed tRNA · TP50·5 S [32P] RNA and tRNA·TP50·t [32P] RNA complexes and investigated the accessibility of the 32P‐labelled tRNAs to ribonuclease T1. It was found that in this complex both 5 S RNA and tRNA are attacked by T1 RNase. In sharp contrast, the addition of 30 S subunit protects 5 S RNA as well as tRNA from degradation. We suggest that 5 S RNA‐TP50 complex is exposed to the ribosomal interface and is involved in subunit interaction.