The proteins of donor tRNA-binding site of Escherichia coli ribosomes

Abstract

Volume 135, number 1 FEBS LETTERS November 1981 THE PROTEINS OF DONOR tRNA-BINDING SITE OF ESCHERICHIA COLI RIBOSOMES S. N. VLADIMIROV, D. M. GRAIFER and G. G. KARPOVA Institute of Organic Chemistry, Siberian Division of the USSR Academy of Sciences, Novosibirsk 630090, USSR Received 1 October 1981 1. Introduction A new photoreactive derivative of tRNA Phe con- taining several arylazido-groups scattered statistically over the tRNA's guanine residues was proposed for photoaffinity labeling of tRNA-binding sites of ribo- somes from Escherichia coli [1,2]. It was obtained in two steps: (i) Statistic alkylation of N7 atoms of guanosine in tRNA by 4-(N-2-chloroethyt-N-methylamino)- benzylamine up to the average extent of modifi- cation 3-4 tool reagent/tool tRNA; (ii) Selective attaching of photoreactive groups to the aliphatic amino groups of reagent residues by treating of alkylated tRNA with 2,4-dinitro-5- fluorophenylazide [1]. This derivative is competent for non-enzymatic, poly(U)-dependent binding with ribosomes and being bound may be crosslinked with ribosome by UV4rra- diation [t,2]. Here, we show this photoreactive derivative of tRNA phe (I) to bind only at the ribosomal P-site in the presence of the excess of ribosomes and poly(U). After UV-irradiation of the ternary complex 70 S. poly(U) - I the derivative of tRNA Phe was cova!ently linked to 30 S and 50 S ribosomal subunits. We have found that the proteins $5, $9, S11, S12, S13, S19 and $21 were labeled in the 30 S subunits and the proteins L11, L13, L14 and possibly L27 in the 50 S ones. No modification of 16 S and 23 S RNAs was observed. 2. Materials and methods Ribosomes were isolated from E. coli MRE-600 as in [3]. 4-(N-2-Chloroethyl-N-methylamino)-[14C] - benzylamine (25 mCi/mmol) was synthesized as in [4,5]. 2,4-Dinitro-5-fluorophenylazide was prepared as in [6]. PolyCC) with av. M r 30 000 was from SCTB of biologically active compounds (USSR). [14C[- Phenylalanine (360 mCi/mmol) was from UVVVR (Czechoslovakia), ATP was from Reanal (Hungary), RNases A and T1 were from Sankyo Co. (Japan) and tRNA Phe was from Sigma. Phenylalanyl-tRNA- synthetase was a kind gift from Dr S. Khodyreva. Aminoacylation of tRNA Phe was done as in [7]. For isolation of Phe-tRNA Phe 1 ml reaction mixture was passed through the column with DEAE (DE-52)-celtu- lose (0.2 ml). The column was washed with 0.2 M NaC1 and then Phe-tRNA Phe was eluted from the DEAE-cellulose with 1 M NaC1. Alkylation of tRNA Phe under the conditions the lability of tRNA tertiary structure and follow- ing attaching of arylazido-groups to the aliphatic amine residues was performed as in [1 ]. The ternary complex 70 S ribosome • poly(U) • Phe-tRNA Phe was obtained in buffer A (0.05 M Tris-HC1 (pH 7.3), 0.1 M NH4C1, 0.02 MgCt2) using 3-5-fold excess of ribosomes to tRNA at 0°C. In the experiments on inhibition of binding of [14C]Phe-tRNAPhe to 70 S. poly(U) complex by unlabeled I both tRNA species were added to the complex simultaneously. In the experiments with tetracycline (Tet) ribosomes were preincubated with Tet (4 × 10 -s M) at 0°C for 30 min. The ternary complex 70 S • poly(U) • I was formed in the buffer A at 0°C for 2 h. Concentrations of I, ribosomes polyCtJ)were 10-6 M, 3.5 × M and 0.1 mg/ml, respectively. To obtain the covalent bond between I and the ribosome the solution con- taining ternary complex was passed through a cuvette cooled to 13°C at a rate of ~2.4 ml/h. The cuvette was irradiated with a high pressure mercury lamp (500 W), X >~ 350 nm (glass and water filters). After irradiation the ribosomes were precipitated by adding 0.8 vol. ethanol and subsequent separation into 30 S Published by Elsevier/North.Holland Biomedical Press 00145793/81[0000-0000[$02.75 © 1981 Federation of European Biochemical Societies 155