A High-Throughput Assay for In Vitro Determination of Release Factor-Dependent Peptide Release from a Pretermination Complex by Fluorescence Anisotropy-Application to Nonsense Suppressor Screening and Mechanistic Studies

Abstract

Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases. We recently elucidated ataluren's mechanism of action as a competitive inhibitor of release factor complex (RFC) catalysis of premature termination and identified ataluren's binding sites on the ribosome responsible for such an inhibition. These results suggest the possibility of discovering new TRIDs, which would retain ataluren's low toxicity while displaying greater potency and generality in stimulating readthrough via the inhibition of termination. Here we present a detailed description of a new in vitro plate reader assay that we are using both to screen small compound libraries for the inhibition of RFC-dependent peptide release and to better understand the influence of termination codon identity and sequence context on RFC activity.

Keywords: ataluren; high-throughput screening (HTS); premature termination codon; readthrough; termination; translation readthrough-inducing drug (TRID).

Figure 1

Competition for binding to a UGA PTC between release factor complex, leading to peptidyl-tRNA hydrolysis and nascent peptide release (i.e., termination), and aminoacyl suppressor tRNA ternary complex, leading to readthrough. In our standard HT assay, Stop-POST5 contains FK*VRQ-tRNA in the P-site, where K* denotes a Lys residue labeled with Atto647 on the ε-amino group.

Figure 2

Measures of pentapeptide release at pH 7.5. In all of the experiments, Stop-POST5, 0.05 µM, was prepared using KCl-treated 80S ribosomes programmed with CrPV-IRES mRNA1, and GTP was present at 1 mM. (A) Release of Atto647-labeled and unlabeled pentapeptides measured by co-sedimentation. Conditions: 10 min incubation, 37 °C, eRF1 0.2 µM, and eRF3 0.4 µM. (B) Rates of Atto647-labeled pentapeptide release measured by co-sedimentation and TLE assays. Conditions: eRF1 0.1 µM, eRF3 0.2 µM, and 25 °C. (C) Rates of dye-labeled pentapeptide release as measured in the plate reader assay using three different dyes. Apparent rate constants were determined by fitting traces to a one phase decay model (Graphpad prism). Conditions: eRF1 0.2 µM, eRF3 0.4 µM, and 25 °C. (D) Rate of unlabeled pentapeptide release measured by Millipore filtration, calculated using both filtrate and filter measurements. Conditions: eRF1, 0.2 μM, eRF3 0.8 μM, and 25 °C. Error bars: A and B, ± average deviations, n= 2; D, ± standard deviations, n = 8 for all points except for the 4 min measurements, for which n = 11.

Figure 3

Optimization of the plate reader termination assay. All determinations were made at 25 °C. Except as otherwise indicated below, Stop-POST5 (0.05 µM) was prepared from KCl-treated 80S ribosomes programmed with CrPV-IRES mRNA1, pH was 7.5 and the GTP was 1 mM. (A) pH-dependent traces. Conditions: Stop-POST5, 0.05 µM, eRF1 0.05 µM, eRF3 0.2 µM, and n = 2. The traces are normalized to pH 7.5, making the smaller anisotropy changes observed at a higher pH more apparent. (B) pH-dependent apparent rate constants in A. (C) Comparison of rates for Stop-POST5 prepared from either 40S + 60S subunits or KCl treated 80S. Conditions: eRF1 0.025–0.40 µM and eRF3 0.8 µM. Error bars are average deviations, n = 2. (D) eRF3 stimulation of eRF1 termination activity, eRF1 and eRF3 concentrations as shown, n = 2. (E) Dependence of termination rate on eRF3 concentration. Conditions: Stop-POST5 0.03 µM, eRF1 0.05 µM, eRF3 0.01–0.1, and n = 2. (F) Comparison of the cold stabilities of yeast and human RFCs. Conditions: eRF1 0.05 µM, eRF3 0.8 µM, and n = 2. (G–J) Effects on termination activity of adding small percentages of the specified water-miscible organic solvents to the reaction medium. Conditions: eRF1 0.05 µM, eRF3 0.1, and n = 2. In each figure, traces are normalized to the trace containing no added solvent, making clear the smaller anisotropy change observed with either DMSO or EtOH.

Figure 4

Applications of the high-throughput peptide release assay. All determinations were performed at 25 °C, pH 7.5, and 1 mM GTP. Stop-POST5 was prepared from KCl-treated 80S ribosomes programmed with either CrPV-IRES mRNA1 (A–E), CrPV-IRES mRNA2 (F), or CrPV-IRES mRNA3 (G). Stop-POST5 was present at either 0.05 µM (A–D) or 0.04 µM (E–G). (A) Sample ataluren inhibition traces. (B) Inhibition as a function of ataluren concentration. Conditions: eRF1 0.0625 µM, eRF3 0.8 µM, and n = 2. (C) Failure of GMQ to inhibit peptide release, 25–200 µM. Conditions: eRF1 0.0625 µM, eRF3 0.8 µM, and n = 2. (D) Inhibition by candidate TRIDs added at 300 µM compared with ataluren added at 500 µM. Conditions: eRF1 0.0625 µM, eRF3 0.8 µM, n = 2. (E–G) Sample results showing strong influence of stop codon (shown in red) and downstream codons on EC₅₀ and V_max values for RFC catalysis of peptide release.