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1.
Figure 4

Figure 4. From: The 2?-OH group of the peptidyl-tRNA stabilizes an active conformation of the ribosomal PTC.

The dA76-substituted aa-tRNA is inherently less reactive than its rA76 counterpart. A graph of the observed rates of background hydrolysis and aminolysis (with Tris and hydroxylamine) in a buffered solution. Points plotted represent rates measured from two independent measurements.

Hani S Zaher, et al. EMBO J. 2011 June 15;30(12):2445-2453.
2.
Figure 1

Figure 1. From: The 2?-OH group of the peptidyl-tRNA stabilizes an active conformation of the ribosomal PTC.

The environment of the active site of the ribosome during peptidyl transfer and peptide release. Structure of the PTC showing the proximity of the 2′-OH group of A76 to the nucleophile and leaving group and to that of A2451 during the course of peptide-bond formation (A) and RF-mediated peptide release (B). Models were constructed using PyMol and PDB files (2WDK, 2WDL, 1VQ7, 2WDG, 2WDI, 2X9R, 2X9S, 3D5A, and 3D5B).

Hani S Zaher, et al. EMBO J. 2011 June 15;30(12):2445-2453.
3.
Figure 6

Figure 6. From: The 2?-OH group of the peptidyl-tRNA stabilizes an active conformation of the ribosomal PTC.

RF-mediated release is inhibited in the absence of the 2′-OH of A76 of the peptidyl-tRNA. An autoradiograph of an electrophoretic TLC used to follow the release of the fMet-Lys dipeptide in the presence of the indicated Lys-tRNALys and RF (oe and ce denote overexpressed and chromosomally expressed factors, respectively). As expected, when the depicted IC is incubated with rA76 Lys-tRNALys, EFG, and any of the RFs, quantitative release of the dipeptide is observed in all cases. In contrast, when the rA76 aa-tRNA is substituted by the dA76 one, efficient release of the dipeptide is only observed in the presence of the rescuing RF1GGS factor.

Hani S Zaher, et al. EMBO J. 2011 June 15;30(12):2445-2453.
4.
Figure 2

Figure 2. From: The 2?-OH group of the peptidyl-tRNA stabilizes an active conformation of the ribosomal PTC.

The donor activity of the dA76-substituted peptidyl-tRNA critically depends on the assay set-up. An autoradiograph of an electrophoretic TLC used to follow tripeptide formation with rA76 and dA76 tRNAs under different conditions. When a Met-Lys-Phe programmed IC (left panel) is incubated with rA76 Lys-tRNALys and Phe-tRNAPhe ternary complexes in the presence of EFG, efficient tripeptide synthesis is observed regardless of the course of ternary complexes addition. In contrast, for the dA76 reaction, no tripeptide synthesis is observed when Phe-tRNAPhe ternary complex is added 5 min subsequent to the addition of the Lys-tRNALys ternary complex and EFG; tripeptide synthesis is only observed when both ternary complexes and EFG are added simultaneously to the IC, albeit with a reduced yield (∼40%). In agreement with these observations, a pelleted rA76 dipeptidyl RNC (right panel) reacts efficiently with the Phe-tRNAPhe, while its dA76 counterpart fails to produce significant amount of the tripeptide.

Hani S Zaher, et al. EMBO J. 2011 June 15;30(12):2445-2453.
5.
Figure 5

Figure 5. From: The 2?-OH group of the peptidyl-tRNA stabilizes an active conformation of the ribosomal PTC.

Defects in the acceptor activity of the dA76 aa-tRNA are the result of poor ternary complex formation. (A) Time courses of dipeptide formation with an IC and Lys-tRNALys tRNA. The aa-tRNA was added to a final concentration of 0.5 μM (half of the IC concentration). For the rA aa-tRNA, the addition of EFTu stimulated the rate of PT by ∼200-fold (∼0.02 versus 4 s−1), while it had no discernible effect on the dA76 reaction (∼0.02 s−1 for both). (B) Time courses of GTP hydrolysis. While the rate of GTP hydrolysis for the rA76 was determined to be ∼10 s−1, no apparent rate could be measured for the dA76 substrate. (C) Time courses of aa-tRNA dissociation from EFTu.GTP using an RNase protection assay. While the rA76 aa-tRNA dissociated at a rate of ∼0.05 s−1, the dA76 aa-tRNA was fully dissociated from EFTu.GTP at the 5-s interval (the lowest time-point), suggesting that it never bound the factor or their interaction is very labile.

Hani S Zaher, et al. EMBO J. 2011 June 15;30(12):2445-2453.
6.
Figure 3

Figure 3. From: The 2?-OH group of the peptidyl-tRNA stabilizes an active conformation of the ribosomal PTC.

Disentangling the effects of the dA76 substitution of the peptidyl-tRNA on the rate of peptide-bond formation. (A) Time courses of tripeptide formation with a dipeptidyl Met-Lys RNC and Phe-tRNAPhe ternary complex. Consistent with the rearrangement mechanism, the rate of peptide-bond formation with the dA76 dipeptidyl RNC could not be measured, while that with the rA76 complex was measured to be 30 s−1. (B) Time courses of dipeptide and tripeptide formation with an IC and simultaneous incubation with Lys-tRNALys and Phe-tRNAPhe ternary complexes. The donor activity of the dA76-substituted peptidyl-tRNA was found to be limited by the acceptor activity of its aa-tRNA equivalent. The observed rate of Met-Lys-Phe tripeptide formation (dA-tri) is similar to that determined for the formation of the Met-Lys dipeptide (dA-di, ∼0.02 s−1), and is about 100-fold slower than that determined for the tripeptide with rA76 aa-tRNA (rA-tri). As seen earlier the yield of tripeptide formation in the presence of the dA76 substrate is ∼50%. (C) Time course of tripeptide formation between a pre-translocation dipeptidyl Met-Lys RNC and Phe-tRNAPhe ternary complex in the presence of EFG. The rate of peptide-bond formation with a dA76-substituted peptidyl-tRNA is reduced by more than two orders of magnitude relative to the rA76 peptidyl-tRNA, while the end point was measured to be 0.4 instead of the 0.8 end point measured for the rA76 substrate.

Hani S Zaher, et al. EMBO J. 2011 June 15;30(12):2445-2453.

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