Progress Towards a Mechanistic Understanding of Peptide Bond Formation

Gregory W. Muth and Scott A. Strobel
Department of Molecular Biophysics and Biochemistry
Yale University, New Haven, CT

The publication of the crystal structure of the 50S ribosomal subunit provided evidence that solely RNA potentially catalyzes the process of peptide bond formation in a process historically referred to as peptidyl transfer. This universal process of peptide synthesis occurs between amino acid bearing tRNA molecules bound in the ribosome coded by mRNA. Over the years in vitro kinetic assays have been developed with clearly defined experimental conditions that show how the ribosome is capable of catalyzing peptide bond formation between two fragmented tRNA molecules.  These "fragment assays" show peptidyl transfer occurring efficiently when only peptidyl CCA or aminoacyl CCA fragments are used in place of full length tRNAs.  The peptidyl CCA fragment, also known as the P-site substrate, consists of an aromatic amino acid linked to the 3'-OH of the ribose sugar through an ester bond.  The alpha-amino nucleophile of the aminoacyl CCA fragment bound in the A-site of the ribosome attacks this ester, thus forming a new amide bond (Figure 1).

Figure 1.  Simplified mechanism of peptidyl transfer within the ribosome.  The A-site aa-tRNA acts as a nucleophile and attacks the carbonyl carbon of the P-site bound tRNA peptide ester.  This reaction results in the formation of the charged tetrahedral intermediate that spontaneously resolves into the newly formed peptide bond and the release of the tRNA hydroxyl leaving group.

It is hypothesized that the RNA within the active site may catalyze peptide bond formation in several ways [1-3].  At physiological pH, the alpha-amino group is protonated thus making it inactive as a nucleophile.  Ribosomal RNA may aid in the deprotonation of this amine [4].  After nucleophilic attack, the charged tetrahedral intermediate and/or leaving group hydroxyl could be stabilized by the 2'-OH of the P-site CCA fragment or other RNA moieties within the active site (Muth et al, in preparation).  Finally, the RNA within the active site may simply provide a scaffolding which orients the fragments in the correct alignment and provides a significant rate enhancement.  To understand how RNA may catalyze peptide bond formation within the active site of 50S ribosomal subunits we have undertaken the synthesis of several new tRNA "fragments" which will allow us to systematically examine the deprotonation of the amine nucleophile, the role of the 2'-OH on the P-site CCA fragment and the absolute stereochemisrty of the tetrahedral intermediate (Figures 2-4).

Figure 2.  Hydroxypuromycin was synthesized to replace the commonly used substrate puromycin (a known antibiotic).  Puromycin and hydroxypuromycin both bind to the ribosomal A-site and act as nucleophiles on the P-site ester. Hydroxypuromycin does not posses a pH sensitive alpha amine group but rather the hydroxyl depicted in blue on the structure.  This difference allowed us to determine that the pH dependence of the peptidyl transfer reaction was due solely to a group within the ribosome itself.


Figure 3.  This rather imposing molecule is one of a series of inhibitors synthesized by solid phase chemistry that allowed a series of questions to be asked regarding the structure and contributions of the active site rRNA.  The use of solid phase synthesis allowed us to easily change the nature of the nucleotides as well as the linkages between them to build a library of compound suitable for testing in our kinetics assay.  Careful examination should reveal the P-site CCA nucleotides joined to the A-site hydroxypuromycin through a chiral phosphorous group.  The chiral phosphorous structure serves as a stable functionality that represents the correct charge and geometry of the tetrahedral intermediate formed during the peptidyl transfer reaction.

Figure 4.  Similar to the structure in Figure 3, this molecule, also synthesized by solid phase chemistry, was used to explore the role of the 2'-hydroxyl in stabilizing the transition state.  The most remarkable aspect of this molecule was the need to completely re-engineer the chemistry used in solid phase synthesis as the ester linkage is easily hydrolysis under the mildly basic conditions used in conventional deprotection reactions.

1. Muth, G.W., L. Ortoleva-Donnelly, and S.A. Strobel, A single adenosine with a neutral pK(a) in the ribosomal peptidyl transferase center. Science, 2000. 289(5481): p. 947-950.

2. Muth, G.W., L. Chen, A.B. Kosek, and S.A. Strobel, pH-dependent conformational flexibility within the ribosomal peptidyl transferase center. RNA-a Publication of the RNA Society, 2001. 7(10): p. 1403-1415.

3. Strobel, S.A., G.W. Muth, and L. Chen, Exploring the mechanism of the peptidyl transfer reaction by chemical footprinting. Cold Spring Harbor Symposia On Quantitative Biology, 2001. 66: p. 109-117.

4. Katunin, V.I., G.W. Muth, S.A. Strobel, W. Wintermeyer, and M.V. Rodnina, Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome. Molecular Cell, 2002. 10(2): p. 339-346.