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

Figures

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  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.

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  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.

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  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:…

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  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 Vmax values for RFC catalysis of peptide release.