Regiospecificity of the Peptidyl tRNA Ester within the Ribosomal P Site

Huang, Kevin S.; Weinger, Joshua S.; Butler, Ethan B.; Strobel, Scott A.

doi:10.1021/ja0554099

Cited by 24 publications

(26 citation statements)

References 16 publications

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“…It seems that at that stage the P-site A76 O2Ј reaches a position allowing the formation of a hydrogen bond within the TS. Such an H bond can stabilize the TS geometry, as recently suggested based on biochemical studies (17), which means that the TS for the peptide bond is made rather early in the rotation. However, the final bonds made and broken, resulting from the TS will achieve their equilibrium values after further rotation along the guiding reaction pathway associated with the 2-fold axis of the PTC.…”

Section: Discussionmentioning

confidence: 69%

The transition state for formation of the peptide bond in the ribosome

Gindulytė

Bashan

Agmon

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Using quantum mechanics and exploiting known crystallographic coordinates of tRNA substrate located in the ribosome peptidyl transferase center around the 2-fold axis, we have investigated the mechanism for peptide-bond formation. The calculation is based on a choice of 50 atoms assumed to be important in the mechanism. We used density functional theory to optimize the geometry and energy of the transition state (TS) for peptide-bond formation. The TS is formed simultaneously with the rotatory motion enabling the translocation of the A-site tRNA 3′ end into the P site, and we estimated the magnitude of rotation angle between the A-site starting position and the place at which the TS occurs. The calculated TS activation energy, E a , is 35.5 kcal (1 kcal = 4.18 kJ)/mol, and the increase in hydrogen bonding between the rotating A-site tRNA and ribosome nucleotides as the TS forms appears to stabilize it to a value qualitatively estimated to be ≈18 kcal/mol. The optimized geometry corresponds to a structure in which the peptide bond is being formed as other bonds are being broken, in such a manner as to release the P-site tRNA so that it may exit as a free molecule and be replaced by the translocating A-site tRNA. At TS formation the 2′ OH group of the P-site tRNA A76 forms a hydrogen bond with the oxygen atom of the carboxyl group of the amino acid attached to the A-site tRNA, which may be indicative of its catalytic role, consistent with recent biochemical experiments.

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Section: Discussionmentioning

confidence: 69%

The transition state for formation of the peptide bond in the ribosome

Gindulytė

Bashan

Agmon

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…3). 31 The reaction involves aminolysis of the P-site ester by the A-site α-amino group. The ribosome aligns the two substrates such that the amine approaches the sc face of the P-site ester, resulting in a chiral transition state with an S stereochemistry.…”

Section: Ribosomal Peptide Bond Formation By Substrate Assistancementioning

confidence: 99%

RNA catalysis: ribozymes, ribosomes, and riboswitches

Strobel

Cochrane

2007

Current Opinion in Chemical Biology

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The catalytic mechanisms employed by RNA are chemically more diverse than initially suspected. Divalent metal ions, nucleobases, ribosyl hydroxyl groups, and even functional groups on metabolic cofactors all contribute in the various strategies employed by RNA enzymes. This catalytic breadth raises intriguing evolutionary questions about how RNA lost its biological role in some cases, but not in others, and what catalytic roles RNA might still be playing in biology.RNA is well-suited for its role as a purveyor of genetic information. The consistent hydrogen bonding potential of each nucleobase is critical for fidelity during replication, transcription and translation. It is easy to imagine evolutionary pressures that would select for residues with such properties. This is achieved in part because none of the nucleobases have ionizable groups near neutrality Although optimized for reliable base pairing, RNA is also able to catalyze essential biochemical reactions, including RNA processing and protein synthesis. It does this despite significant biophysical handicaps. RNA has a small repertoire of functional groups and these are embedded in a poorly-constrained ribosyl-phosphate backbone heavy with negative charge. The pK a s of the bases would appear to be either too low (3.5 for A, 4.2 for C) or too high (9.8 for G, 10.5 for U) for use in efficient general acid or base catalysis. As a group, RNA enzymes, or ribozymes, are less efficient catalysts relative to chemically supercharged proteins, yet in many cases ribozymes provide enough rate enhancement to have escaped replacement by protein alternatives in the evolution from an RNA World to the protein dominated world of modern biochemistry.In this review we will examine four examples of RNA catalysis, each of which provides a different chemical strategy used to promote a ribozyme reaction. This includes: the group I class of self-splicing introns which employs two divalent metal ions to promote RNA cleavage and ligation reactions; the Hepatitis Delta Virus ribozyme which catalyzes autolytic RNA cleavage using an essential, pK a perturbed cytidine; the glmS ribozyme which is an autolytic riboswitch that uses the small molecule metabolite glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor; and the ribosomal peptidyl transferase center which doesn't appear to use any of these strategies, but instead forms peptide bonds with the assistance of a functional group on the tRNA substrate. The diversity of solutions employed by RNA to achieve reaction catalysis is unexpected; raising intriguing questions about the evolution and devolution of RNA catalysis. What led RNA to loose its catalytic role in some biological cases but not others, and what other catalytic functions RNA might still be playing in biology?

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“…The organic layer was separated, washed twice with saturated NaHCO 3 -H 2 O (50×2 mL), and dried (MgSO 4 ). Volatiles were evaporated under vacuum, and the resulting mixture was chromatographed (EtOAc/hexanes, 3:7) to give compound 11 (0.96 g, 84%): 1 H NMR (500 MHz, DMSO-d 6 …”

Section: -N-benzoyl-2'-o-t-butyldimethylsilyl-5'-o-(44'-dimethoxytrmentioning

confidence: 99%

“…5 The ribosome aligns the two substrates such that reaction proceeds through a chiral transition state with an S stereochemistry. 6 This configuration places the critical A76 2'-OH between the α-amino nucleophile and the 3'-O leaving group. Possible roles for this hydroxyl in proton transfer from the α-amine to the 3'-O have been proposed.…”

Section: Introductionmentioning

confidence: 99%

“…The 2'-OH vicinal to the P-site ester is essential for the reaction. Deletion or alteration of this functional group results in a complete loss of peptidyl transferase activity (>10 6 fold loss of activity). 4 This contribution is significantly greater than that made by any of the rRNA nucleotides within the active site, though the 2'-OH of A2451 also makes an important contribution.…”

Section: Introductionmentioning

confidence: 99%

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Synthesis of Isotopically Labeled P-Site Substrates for the Ribosomal Peptidyl Transferase Reaction

Zhong

Strobel

2007

J. Org. Chem.

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Isotopomers of the ribosomal P-site substrate, the trinucleotide peptide conjugate CCA-pcb, 1 have been designed and synthesized in 26-350020steps. These include individual isotopic substitution at the α-proton, carbonyl carbon, and carbonyl oxygen of the amino acid, the O2' and O3' of the adenosine, and a remote label in the N3 and N4 of both cytidines. These isotopomers were synthesized by coupling cytidylyl-(3'5')-cytidine phosphoramidite isotopomers, as the common synthetic intermediates, with isotopically substituted A-Phe-cap-biotin (A-pcb). The isotopic enrichment is higher than 99% for 1-13 C (Phe), 2-2 H (Phe), and 3,4-15 N 2 (cytidine), 93% for 2'/3'-18 O (adenosine), and 64% for 1-18 O (Phe). A new synthesis of highly enriched [1-18 O 2 ] phenylalanine has been developed. The synthesis of [3'-18 O] adenosine was improved by Lewis acid aided regioselective ring opening of the epoxide and by an economical S N 2-S N 2 method with high isotopic enrichment (93%). Such substrates are valuable for studies of the ribosomal peptidyl transferase reaction by complete kinetic isotope effect analysis and of other biological processes catalyzed by nucleic acid related enzymes, including polymerases, reverse transcriptases, ligases, nucleases, and ribozymes.

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Regiospecificity of the Peptidyl tRNA Ester within the Ribosomal P Site

Cited by 24 publications

References 16 publications

The transition state for formation of the peptide bond in the ribosome

The transition state for formation of the peptide bond in the ribosome

RNA catalysis: ribozymes, ribosomes, and riboswitches

Synthesis of Isotopically Labeled P-Site Substrates for the Ribosomal Peptidyl Transferase Reaction

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