Stop Codon Recognition by Release Factors Induces Structural Rearrangement of the Ribosomal Decoding Center that Is Productive for Peptide Release  Elaine M. Youngman, Shan L. He, Laura J. Nikstad, Rachel Green  Molecular Cell  Volume 28, Issue 4, Pages 533-543 (November 2007) DOI: 10.1016/j.molcel.2007.09.015 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 The Effect of Mutations in the Ribosomal Decoding Center on Peptide Release (A) Structure of the ribosomal decoding center, showing A1492 and A1493 in their intrahelical h44 positions in the absence of bound substrate (left) and in their “induced” positions in the presence of bound mRNA and a cognate tRNA anticodon stem-loop (ASL) (center), or in the presence of paromomycin and streptomycin (right). Figure was made using PDB entries 1FJF, 1IBM, and 1FJG (Carter et al., 2000; Ogle et al., 2001; Wimberly et al., 2000) and PyMOL (http://pymol.sourceforge.net). (B–E) Rate constants for catalysis at saturating release factor concentrations (kcat) for wild-type and mutant ribosomes on (B) stop- or (C–E) near-stop-programmed ribosomes. Bars represent the mean ± standard error from at least two experiments measured at the following concentrations of RF1: 5 μM for UAG, 125 μM for CAG, 100 μM for UCG, and 200 μM for UAC. Throughout the figures, black bars represent release reactions on stop codons while gray bars indicate reactions on near-stop codons. Molecular Cell 2007 28, 533-543DOI: (10.1016/j.molcel.2007.09.015) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 The Effect of Deoxyribonucleotides in the A Site Codon on Peptide Bond Formation and Peptide Release (A) Rate constants for peptide bond formation on a ribose (filled triangles) or deoxyribose (open triangles) UUU (Phe) codon. On the ribose codon, fitting the data to a hyperbola yields a rate constant of 8.5 ± 1.3 s−1 at saturation. On the deoxyribose codon, the reaction is saturated by 0.5 μM ribosomes and gives a rate constant of (1.4 ± 0.08) × 10−4 s−1. (B) Reaction curves at saturating RF concentrations for peptide release on a ribose (filled triangles) or deoxyribose (open triangles) UAA (stop) codon. Fitting to a single exponential gives a rate constant of 0.34 ± 0.03 s−1 for the ribose codon, and 0.25 ± 0.004 s−1 for the deoxyribose codon. RF concentration was 5 μM for ribose, 10 μM for deoxyribose. Error bars represent standard error from at least two experiments. Molecular Cell 2007 28, 533-543DOI: (10.1016/j.molcel.2007.09.015) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 The Effect of Aminoglycosides on Peptide Release Observed rate constants for peptide release on the stop codon UAA or three near-stop codons in the absence or presence of (A) streptomycin or (B) paromomycin. In every case, rate constants were measured at 5× the K1/2 of RF1 in the absence of antibiotic (Freistroffer et al., 2000). Those concentrations are as follows: 40 nM for UAA, 100 μM for CAA, and 50 μM for UCA and UAC. Streptomycin was present at 20 μM. Paromomycin was used at 50 μM for UAA and 5 μM for all other codons. Bars represent the mean ± standard error from at least two experiments. Molecular Cell 2007 28, 533-543DOI: (10.1016/j.molcel.2007.09.015) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 The Kinetic Mechanism of Paromomycin Inhibition Is Different on Stop and Near-Stop Codons (A–C) Paromomycin is a competitive inhibitor of RF binding to ribosomes programmed with a stop codon. (A) The K1/2 of RF1 for stop codon-programmed complexes increases with increasing paromomycin concentration. At each concentration of paromomycin, RF1 titrations were performed and the resulting kobs versus [RF1] curves were fit to a hyperbolic equation. The bars represent the best-fit K1/2 values ± the error in the fit. (B) The IC50 of paromomycin for the release reaction on stop codon-programmed ribosomes increases with increasing RF1 concentration. IC50 values were determined by performing paromomycin titrations at a given concentration of RF1 and fitting the resulting kobs versus [paromomycin] curves to a hyperbolic equation. Bars represent the best-fit IC50 values ± the error in the fit. (C) The rate constant for peptide release at saturating RF1 concentrations does not change in the presence of increasing concentrations of paromomycin. Bars represent the mean kcat ± standard error from at least two experiments. (D and E) Paromomycin affects both K1/2 and kcat values for peptide release on near-stop codon-programmed ribosomes. (D) K1/2 and (E) kcat values in the absence (open bars) or presence (filled bars) of 5 μM paromomycin. RF1 titrations were performed, and the resulting kobs versus [RF1] curves were fit to a hyperbolic equation. Bars represent the best-fit values ± the standard error in the fit. Molecular Cell 2007 28, 533-543DOI: (10.1016/j.molcel.2007.09.015) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Decoding Site Mutations and Paromomycin Act Synergistically to Inhibit Peptide Release (A) kcat and (B) K1/2 values for peptide release on the stop codon UAG by decoding site variant ribosomes in the absence (open bars) and presence (closed bars) of 25 μM paromomycin. kcat values are the mean ± standard error from 2 measurements of the rate constant for peptide release at saturation (5 μM RF1 in the absence or 150 μM RF1 in the presence of paromomycin). K1/2 values were determined as in Figure 4 and bars represent the best-fit values ± the standard error in the fit. Molecular Cell 2007 28, 533-543DOI: (10.1016/j.molcel.2007.09.015) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 RF1 and Paromomycin Binding Are Mutually Exclusive on Ribosomes Programmed with Stop, but Not with Near-Stop, Codons (A and B) Primer extension on 16S rRNA from (A) UAA- or (B) UCA-programmed ribosome complexes modified with DMS in the presence or absence of paromomycin and RF1. G, C, and A are dideoxy sequencing lanes. Arrow, A1408. (C) Quantitation of the data in (A) and (B). A1408 band intensity was first normalized to a constant band in each lane (arrowhead). The data are presented as normalized A1408 intensity in the presence of paromomycin, with 1.0 defined as the A1408 band intensity under the same conditions in the absence of paromomycin. Molecular Cell 2007 28, 533-543DOI: (10.1016/j.molcel.2007.09.015) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Model for the Accuracy of Stop Codon Recognition (A) When RF1 binds to ribosomes with a stop codon in the A site, a change in the conformation of the termination complex is induced that results ultimately in an “induced” ribosome state that is fully active for peptide release. This state includes a conformation of the decoding center that is not compatible with paromomycin binding. (B) When RF1 binds to ribosomes with a near-stop codon in the A site, it is unable to induce the conformational change that leads to fast peptide release. In the context of this “uninduced” decoding site conformation, paromomycin remains bound and further inhibits peptide release. Molecular Cell 2007 28, 533-543DOI: (10.1016/j.molcel.2007.09.015) Copyright © 2007 Elsevier Inc. Terms and Conditions