Studies on Transfer Ribonucleic Acid-Ribosome Complexes

Peptidylpuromycin formation on mammalian polysomes. Transpeptidation and translocation

“…Gougerotin and relatively high concentrations of cycloheximide and fusidic acid are not competitive. Similar results were obtained by Pestka et al (1973) for anisomycin, sparsomycin, gougerotin, and cycloheximide in an assay essentially the same as that used here. The previously observed effects of these antibiotics on ribosomal function have been reviewed recently (Pestka, 1971a).…”

“…If this is the case, the extent of the reaction is not directly a measure of the total number of available reactive polypeptide chains, but is rather a reflection of the initial rate of the reaction. The Km for puromycin in transpeptidation on rat liver polyribosomes based on initial rates determined under somewhat different conditions from those used in these experiments is 8 X 10-6 m (Pestka et al, 1972). Calculation of a "Km" for puromycin substituting the final values in Figure 3 for initial rates gives a comparable value of 2 X 1CF5 m. The initial rate, and in this case the final value, would be influenced by the concentration of translocated peptide chains as well as by that of puromycin.…”

“…scribed here. Our present studies were carried out before we were aware of the superiority of K+ over NH4+ (Pestka et al, 1972) for the system, but preliminary evidence suggests that substitution of K+ would not alter our general conclusions. The reaction of nascent polypeptide chains to form peptidyl['H]puromycin in the absence of EF-2 is linear with time for at least 3 min, a period sufficiently long to permit kinetic studies.…”

“…Thus, we studied the effects of some inhibitors of protein synthesis on transpeptidation. Recently, Pestka and his collaborators2 (Pestka, 1972;Pestka et al, 1972Pestka et al, , 1973 have used a similar assay with both bacterial and mammalian ribosomes to elucidate some of the basic characteristics of transpeptidation. They have also studied the effect of inhibition of the process in much the same way as de- vs. puromycin concentrations.…”

An Inhibitor of Ribosomal Peptidyl Transferase Using the Transition-State Analogy

“…It has been argued that simply positioning the reacting amino and ester groups for addition could account for the observed reaction rate (Nierhaus et al, 1980). However, a chemical involvement of peptidyl transferase in catalysis is possible (Pestka et al, 1972;Nierhaus et al, 1980). For example, reaction may be accelerated by deprotonation of the attacking amino group and/or stabilization of the transitional oxyanion.…”