Ribosome‐Catalyzed Peptidyl Transfer

Effects of cations and pH value on ribosome‐catalyzed peptidyl transfer were studied, using as assays the reaction of puromycin with (a) washed, polyphenylalanine‐charged Escherichia coli ribosomes and (b) terminal fragments from formlyl‐methionyl‐tRNA (CAACCA‐Met‐F and CCA‐Met‐F) in the presence of purified 50 S ribosomal subunits. In both systems the reaction required the presence of suitable divalent and monovalent cations, and was progressively inhibited below pH 8.5. In the polyphenylalanine system Mg 2+ was active over the range 0.3 mM to 1 M, with a broad optimum from approximately 3 to 100 mM. Mg 2+ was replaceable by Ca 2+ or Mn 2+ . Other divalent metal ions were inert or inhibitory. Spermidine was also inert. Monovalent cations were active in the order, NH 4+ > K + = Rb + > Cs + . NH 4 + and K + were progressively more effective from 10 mM to at least 1 M. Na + and Li + were inert. The reaction of formyl‐methionyl oligonucleotides with puromycin showed similar responses to the polyphenylalanine system except that (a) Mg 2+ was active over the range 5 mM to 1 M with an optimum at approximately 100 mM, (b) Ca 2+ was inert, (c) K + was inactive below 100 mM, and (d) NH 4 + was only weakly active. The bearing of the results on the mechanism of ribosome‐catalyzed peptidyl transfer is discussed, and a possible application of the results is noted.