Different aa-tRNAs Are Selected Uniformly on the Ribosome Sarah Ledoux, Olke C. Uhlenbeck  Molecular Cell  Volume 31, Issue 1, Pages 114-123 (July 2008) DOI: 10.1016/j.molcel.2008.04.026 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Secondary Structures of Ten E. coli aa-tRNAs Used for This Study Anticodon nucleotides are circled, and posttranscriptionally modified nucleotides are in bold and abbreviated according to Sprinzl et al. (1998). Molecular Cell 2008 31, 114-123DOI: (10.1016/j.molcel.2008.04.026) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Key Steps in the Decoding Pathway Molecular Cell 2008 31, 114-123DOI: (10.1016/j.molcel.2008.04.026) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Binding of aa-tRNA to the Entry Site of E. coli 70S Ribosomes Representative equilibrium binding curve for Val-tRNAGACVal·EF-Tu(H84A)·GTP ternary complex to programmed ribosomes in RB buffer at 20°C. The curve corresponds to a single binding isotherm with Kd = 0.93 nM and a fraction bound at saturation of 68%. Ternary complex containing Val-tRNAGACVal (●), Val-tRNAGACVal with no EF-Tu·GTP present (■), ternary complex containing Val-tRNAGACVal with a mRNA containing a noncognate AAA codon (♦) and with no mRNA present (▴). Molecular Cell 2008 31, 114-123DOI: (10.1016/j.molcel.2008.04.026) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Rate of Ribosome-Stimulated GTP Hydrolysis (A) Representative time course for GTP hydrolysis with 300 nM ternary complex containing Val-tRNAGACVal, 3.5 μM encoded ribosomes in RB buffer at 20°C. The data are described by a single exponential fit with kGTP apparent = 30 s−1. (B) Ribosome saturation curve for Val-tRNAGACVal fit to a single Michaelis Menten expression with kGTP at saturation = 38 ± 4.6 s−1. Molecular Cell 2008 31, 114-123DOI: (10.1016/j.molcel.2008.04.026) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Observed Rate of Formation of the Peptide Bond (A) Assay employs an aa-tRNA that is 32P-labeled at the terminal adenosine and loaded into the A site by EF-Tu·GTP. After Val-tRNAGACVal reaction with fMet-tRNAfMet in the P site, products are cleaved by S1 nuclease (arrow) to release valyl-AMP, fMet-valyl-AMP, and free AMP, which are separated by TLC. (B) Representative data for accommodation assay performed with a 3′ 32P-labeled Val-tRNAGACVal (●, kpep = 1.4 s−1, raw data shown in [A]) or a 35S-labeled P site fMet-tRNAfMet (□, kpep = 1.8 s−1) in RB buffer at 20°C fit to a single exponential curve. Data are normalized to 100% dipeptide formation at the final time point of each reaction. Molecular Cell 2008 31, 114-123DOI: (10.1016/j.molcel.2008.04.026) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Summary of Decoding Properties of Ten aa-tRNAs A 2-fold range of the overall mean value for each step is shown as a horizontal shaded bar. Anticodons for each aa-tRNA are shown in parentheses. Molecular Cell 2008 31, 114-123DOI: (10.1016/j.molcel.2008.04.026) Copyright © 2008 Elsevier Inc. Terms and Conditions