Influence of temperature and monovalent cations on reactivity of the donor and acceptor sites on mammalian ribosomes

Abstract

Rat liver ribosomes were prepared with the peptidyl-tRNA either predominantly at the acceptor site (acceptor ribosomes) or at the donor site (donor ribosomes) and were washed free of elongation factors. When incubated at low concentrations of puromycin in the absence of monovalent salts, the donor ribosomes had little capacity to form a peptidyl derivative with puromycin at 0 °C but became reactive at 37 °C Acceptor ribosomes showed little reactivity towards puromycin at either temperature, thus indicating that they do not undergo significant translocation in media lacking monovalent cations. Incubation in the presence of increasing concentrations of NH 4Cl raised the reactivity with puromycin of both donor and acceptor ribosomes to a maximum at 160 mM concentration. The acceptor ribosomes responded to NH 4Cl proportionally more than the donor ribosomes which however still remained more active. This indicates that optimal levels of NH 4Cl can cause the acceptor sites on some but not all acceptor ribosomes to become available to puromycin, presumably by translocation of peptidyl-tRNA from the acceptor site. The increase in reactivity of the fully translocated donor ribosomes shows that NH 4Cl causes changes additional to translocation. Addition of increasing amounts of KCl to the medium also raised the capacity of donor and acceptor ribosomes to react with puromycin, but in this case reactivity of the acceptor ribosomes at 37 °C became indistinguishable from that of donor ribosomes in media containing more than 100 mM KCl, implying complete translocation above this salt concentration. The effect of temperature and salt concentration on binding of aminoacyl-tRNA to the acceptor site was also examined. Raising the concentrations of KCl from 50 to 150 mM increased binding of aminoacyl-tRNA to the acceptor ribosomes, but did not alter binding to donor ribosomes. Higher concentrations of KCl reduced the binding capacity of both types of ribosome. Binding of aminoacyl-tRNA to each ribosome type was temperature-dependent. It is concluded that temperature and the monovalent salt content of the medium play a part in non-enzymic translocation of peptidyl-tRNA from the acceptor to the donor site on mammalian ribosomes, and thus can change the capacity of a population containing acceptor ribosomes to react with puromycin and with aminoacyl-tRNA. In addition, temperature and salt concentration also affect reactivity of donor ribosomes with puromycin, thus indicating that factors other than translocation are involved in the promotion of puromycin peptide formation by monovalent salts. It is suggested that the use of authentic acceptor and donor ribosomes under defined conditions of salt and temperature can increase the information to be learned from reaction with puromycin.