The puromycin reaction mediated by yeast ribosomes in high salt

Abstract

The extend of the reaction between puromycin and yeast peptidyl-tRNA prelabeled in vitro was determined by measuring the distribution of trichloroacetic acid precipitable material in isokinetic sucrose gradients in the presence of 0.5 M KCl.Thus it was found that increasing amounts of puromycin remove increasing amounts of peptidyl-tRNA from the 80S position in the gradient. The extend of the reaction, however, was independent of pretreatment of the ribosomes with inhibitors of the translocation indicating that peptidyl-tRNA at the donor and at the acceptor site of the ribosomes are equally accessible to puromycin at 0.5 M KCl.The exposure of both ribosomal binding sites to puromycin in high salt is accompanied by an enhanced reactivity of puromycin towards peptidyl-tRNA. The ED50 determined by measuring the inhibition by puromycin of the poly-U dependent phenylalanine incorporation drops from 5×10(-5) M below 250 mM KCl to 5×10(-6) M at 300 mM and higher concentrations of KCl.

PURE mRNA display and cDNA display provide rapid detection of consensus binding motif via high-throughput sequencing

“…Next, various salt mix concentrations were tested during post translation incubation. Previously, a high concentration of KCl has been shown to improve the accessibility of ribosomebound peptidyl-tRNA to puromycin (Van Der Mast & Bloemers, 1973) and has been adopted in the recent mRNA and cDNA display methods (Naimuddin & Kubo, 2016;Seelig, 2011;Yamaguchi et al, 2009). We found that without salt addition, conjugate formation is less than 10%, however the addition of 32.5 mM MgCl 2 and 375 mM KCl greatly increased the formation rate to above 40% ( Figure 2B).…”

PURE mRNA display and cDNA display provide rapid detection of core epitope motif via high‐throughput sequencing

“…Translation time reflects the ribosome loading and translation of the coding region which ends up stalling at the 3' end due to lack of stop codon. Previously, a high concentration of KCl has been shown to improve the accessibility of ribosome‐bound peptidyl‐tRNA to puromycin (Van Der Mast & Bloemers, 1973) and has been adopted in the recent mRNA and cDNA display methods (Naimuddin & Kubo, 2016; Seelig, 2011; Yamaguchi et al, 2009). Hence, we tested various salt concentration and found that without salt addition, conjugate formation is less than 10%, however, the addition of 375 mM KCl and 32.5 mM MgCl 2 greatly increased the formation rate to above 40% (Figure 2a).…”