Progress Towards a Mechanistic Understanding of Peptide
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 . 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
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.
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
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):