Accurate Translocation of mRNA by the Ribosome Requires a Peptidyl Group or Its Analog on the tRNA Moving into the 30S P Site  Kurt Fredrick, Harry F Noller  Molecular Cell  Volume 9, Issue 5, Pages 1125-1131 (May 2002) DOI: 10.1016/S1097-2765(02)00523-3

Figure 1 Toeprints Produced by Ribosome Complexes Containing Deacylated tRNA before and after EF-G-Catalyzed Translocation Reactions were processed as described in the Experimental Procedures unless specifically noted. (A) Sequence of message m291, a derivative of the phage T4 gene 32 mRNA, with the relevant P codons marked by braces and their corresponding toeprints indicated by arrows. The first nucleotide of codon 1 (position +1) is shown in bold type, and the Shine-Dalgarno sequence is boxed. (B) Monitoring translocation of codon 2 (UUU) of m291 to the 30S P site within ribosomes. First, ribosomes (0.2 μM) were incubated with m291 (0.4 μM) and tRNAfMet (0.3 μM) to bind the P site, and an aliquot was removed to ice for subsequent extension (P lane). Next, tRNAPhe (0.4 μM) was added to bind the A site, and an aliquot was removed to ice for subsequent extension (A lane). Finally, a volume of the reaction was diluted 2-fold into buffer containing either GTP or EF-G plus GTP, and aliquots were removed after 10 min (−G and +G lanes, respectively). All aliquots were extended in parallel (Joseph and Noller, 1998), and the products were resolved by denaturing PAGE. The open arrowhead indicates a tRNA-independent primer extension product. (C) Monitoring translocation of codon 3 (GUA) of m291 (+SD) or its derivative lacking a Shine-Dalgarno site (−SD) to the 30S P site within ribosomes. The −SD mRNA contains cytosines in place of the two guanines within the boxed sequence shown in (A). In this experiment, tRNAPhe (2 μM) was incubated with ribosomes (1 μM) to fill the P site (P lanes), tRNAVal1 (2 μM) was subsequently added to fill the A site (A lanes), and the reactions were then diluted by 20% into buffer containing GTP (−G lanes) or EF-G plus GTP (+G lanes). Molecular Cell 2002 9, 1125-1131DOI: (10.1016/S1097-2765(02)00523-3)

Figure 2 Monitoring Translocation of Codon 3 (GUA) of m291 with Acylated Forms of tRNAVal1 Pretranslocation complexes were made on m291 by incubating ribosomes with either tRNAPhe or N-acetyl-Phe-tRNAPhe to fill the P site (P lanes) and subsequently adding tRNAVal1, N-acetyl-Val-tRNAVal1, or Val-tRNAVal1 to fill the A site (A lanes) as indicated. Complexes were then diluted by 20% into buffer containing GTP (−G lanes) or EF-G plus GTP (+G lanes). The percentage of posttranslocation complexes correctly positioned with codon 3 (GUA) in the P site (% accuracy) was determined by quantifying the intensities of toeprints at +22 and +18 in the +G lanes, subtracting background from the corresponding −G lanes, and calculating where % accuracy = (+22)/[(+22)+(+18)] × 100%. Overall translocation was estimated from each +G lane by quantifying the intensity of toeprints corresponding to the residual pretranslocation complexes (+19 and +20), quantifying the intensities of all bands from +18 to +22, and calculating where % translocation = {1 − [(+19)+(+20)]/[(+18)+(+19)+(+20)+(+21)+(+22)]} × 100%. Reported values are the mean ± standard deviation of at least three independent experiments (n). Molecular Cell 2002 9, 1125-1131DOI: (10.1016/S1097-2765(02)00523-3)

Figure 3 Monitoring Translocation of Codon 2 (UAC) of m293 with Acylated Forms of tRNATyr2 (A) Sequence of m293, another derivative of the phage T4 gene 32 mRNA, annotated as in Figure 1. (B) Pretranslocation complexes were made on m293 by incubating ribosomes with tRNAfMet to fill the P site (P lanes) and subsequently adding tRNATyr2, N-acetyl-Tyr-tRNATyr2, or Tyr-tRNATyr2 to fill the A site (A lanes) as indicated. Complexes were then diluted by 20% into buffer containing GTP (−G lanes) or EF-G plus GTP (+G lanes). The percentage of posttranslocation complexes correctly positioned with codon 2 (UAC) in the P site (% accuracy) was determined by quantifying the intensities of toeprints at +19 and +15 in the +G lanes, subtracting background from the corresponding −G lanes, and calculating where % accuracy = (+19)/[(+19)+(+15)] × 100%. Overall translocation was estimated from each +G lane by quantifying the intensity of toeprints corresponding to the residual pretranslocation complexes (+16 and +17), quantifying the intensities of all bands from +15 to +19, and calculating where % translocation = {1 − [(+16)+(+17)]/[(+15)+(+16)+(+17)+(+18)+(+19)]} × 100%. Reported values are the mean ± standard deviation of at least three independent experiments (n). Molecular Cell 2002 9, 1125-1131DOI: (10.1016/S1097-2765(02)00523-3)

Figure 4 Monitoring Translocation of Codon 2 (UAC) of m290 with Acylated Forms of tRNATyr2 (A) Sequence of m290, a derivative of the mRNA encoding S21, annotated as in Figure 1. (B) Pretranslocation complexes were made on m290 by incubating ribosomes with tRNAfMet to fill the P site (P lanes) and subsequently adding tRNATyr2, N-acetyl-Tyr-tRNATyr2, or Tyr-tRNATyr2 to fill the A site (A lanes) as indicated. Complexes were then diluted by 20% into buffer containing GTP (−G lanes) or EF-G plus GTP (+G lanes). The percentage of posttranslocation complexes correctly positioned with codon 2 (UAC) in the P site (% accuracy) and the percentage of messages translocated (% translocation) were calculated as described in the legend to Figure 3. Open arrowheads indicate tRNA-independent primer extension products. Molecular Cell 2002 9, 1125-1131DOI: (10.1016/S1097-2765(02)00523-3)