Implication of Arginyl Residues in mRNA Binding to Ribosomes

Abstract

Modification of Esclzevichia coli ribosomes with phenylglyoxal and butanedione, protein reagents specific for arginyl residues, inactivates polypeptide polymerization, assayed as poly(U)-dependent polyphenylalanine synthesis, and the binding of poly(U). Inactivation is produced by modification of the 30-S subunit. Both the RNA and the protein moieties of 30-S subunits are modified by phenylglyoxal, and modification of either of them is accompanied by inactivation of polypeptide synthesis. Modification of only the split proteins released from 30-S subunits by prolonged dialysis against a low-ionic-strength buffer, which contain mainly protein S1, produces inhibition of poly(U) binding and inactivation of polypeptide synthesis. Amino acid analysis of the modified split proteins showed a significant modifications of arginyl residues. These results indicate that the arginyl residues of a few 30-S proteins might be important in the interaction between mRNA and the 30-S subunit, which agrees with the general role assigned to the arginyl residues of proteins as the positively charged recognition site for anionic ligands.The chemical modification of the amino acid residues of proteins with specific reagents has been extensively used to study the active sites of enzymes and the relationships between structure and function in proteins. Treatment of enzymes that bind anionic substrates or cofactors with reagents specific for arginine is frequently accompanied by an inactivation which is correlated with the loss of the capacity for binding the negatively charged ligand [l]. These results indicate that arginyl residues play a general role in the binding of anionic ligands, containing phosphate or carboxylate moieties, by providing a positively charged recognition site [I 1. Since ribosomes bind mRNA and aminoacyl-tRNA during protein synthesis, and these molecules are negatively charged, arginyl residues might also play a fundamental role in their binding.We have studied the effects of modification of ribosomes with the arginine reagents phenylglyoxal and butanedione on poly(U)-directed polyphenylalanine synthesis and poly(U) binding. The results seem to indicate that arginyl residues are implicated in the binding of mRNA to the 30-S ribosomal subunit. MATERIALS AND METHODS MuterialsRibosomes and ribosomal subunits were prepared from Esclzerichiu coli MRE 600 as described previously [2]. Split proteins from 30-S subunits were obtained by dialysis of the subunits against 1 mM Tris-HC1 (pH 7.8) and 10 mM MgC12, and separation of the released proteins by centrifugation [3]. 16-S RNA and total proteins were obtained from 30-S subunits according to Traub el al. [4]. Phenylglyoxal and butanedione were obtained from ECA-Chemie (Steinheim/Albuch). The radioactive compounds mere purchased from the Radiochemical Centre (Amcrsham). Modiji'cation w,itlz Plzenylglyoxul and ButunedionePrior to modification, 70-S ribosomes, ribosomal subunits and split proteins were dialyzed against 100 mM NaHC03 (pH 8.0) and 10 mM MgC12 (modification with ...