Studies on the binding of acylaminoacyl-tRNA to rat liver 60S ribosomal subunits and its participation in the peptidyltransferase reaction.
Peptidyltransferase with rat liver 60S subunits can be measured by the reaction between exogenous acylaminoacyl-tRNA and puromycin to form acylaminoacylpuromycin in the presence of 33% methanol, 0.3 M KCl, and 4 mM MgCl2. An assay system has been developed that allows examination of the binding of acetylphenylalanyl-tRNA to the ribosomal subunit "P" site, the transpeptidation of the 60S-bound substrate to puromycin, and the requirements for these individual steps. Binding of acetylphenylalanyl-tRNA to 60S subunits is stimulated several-fold by the addition of methanol, but the extent of binding in alcohol is the same in 60 as in 300 mM KCl containing solutions. Formation of acetylphenylalanyl-puromycin from 60S-acetylphenylalanyl-tRNA complex and puromycin stringently requires alcohol and the initial rate of the reaction is markedly greater at 300 mM KCl than at 60 mM KCl concentrations. Thus, alcohol and high concentrations of monovalent cation affect the reaction of an event subsequent to the binding of substrate to the "P" site. Preincubation of 60S subunits with poly(U), which stimulates the overall peptidyltransferase reaction, does not affect the amount of acetylphenylalanyl-tRNA that is bound to the particles; however, it markedly stimulates the initial rate of the transpeptidation reaction between 60S-acetylphenylalanyl-tRNA complex and puromycin. The codon specificity and the failure to affect binding with poly(U) suggest a role for the polynucleotide in the alignment or stabilization of the acylaminoacyl-tRNA on the "P" site rather than an effect on binding to either of the two particle sites or on the peptidyltransferase "active center." The effect of 40S subunits, which inhibit the overall peptidyltransferase reaction, on the binding of substrate could not be clearly interpreted since all three preparations, 60S subunits, 40S subunits, and combinations of 60S plus 40S particles, appear to bind acetylphenylalanyl-tRNA in the presence of methanol. However, the initial rate of peptide bond formation is several times greater with 60S-acetylphenylalanyl-tRNA complex than with 60S plus 40S particles containing bound acetylphenylalanyl-tRNA and the addition of 40S subunits to preformed 60S-acetylphenylalanyl-tRNA complex during the transpeptidation phase of the reaction in methanol does not affect the rate of peptide bond formation. Thus, 40S subunits seem to inhibit peptidyltransferase by forming less reactive particles in aqueous solutions. Contd.
D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D000432:Methanol / Q000494:pharmacology; D010455:Peptides; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism*; D051381:Rats; D011955:Receptors, Drug; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*
D010455:Peptides; D011955:Receptors, Drug; D012343:RNA, Transfer; D000217:Acyltransferases; D000432:Methanol