Structural Insights into the Roles of Water and the 2′ Hydroxyl of the P Site tRNA in the Peptidyl Transferase Reaction

Abstract

Peptide bond formation is catalyzed at the peptidyl transferase center (PTC) of the large ribosomal subunit. Crystal structures of the large ribosomal subunit of Haloarcula marismortui (Hma) complexed with several analogs that represent either the substrates or the transition state intermediate of the peptidyl transferase reaction show that this reaction proceeds through a tetrahedral intermediate with S chirality. The oxyanion of the tetrahedral intermediate interacts with a water molecule that is positioned by nucleotides A2637 (E. coli numbering, 2602) and (methyl)U2619(2584). There are no Mg2+ ions or monovalent metal ions observed in the PTC that could directly promote catalysis. The A76 2' hydroxyl of the peptidyl-tRNA is hydrogen bonded to the alpha-amino group and could facilitate peptide bond formation by substrate positioning and by acting as a proton shuttle between the alpha-amino group and the A76 3' hydroxyl of the peptidyl-tRNA.

Cross-crystal averaging reveals that the structure of the peptidyl-transferase center is the same in the 70S ribosome and the 50S subunit

“…Insights into the structural basis of mRNA decoding have come largely from structures of the Thermus thermophilus 30S ribosomal subunit and its substrate complexes (1)(2)(3)(4), whereas understanding of the structural basis of peptide bond formation has been derived from structures of the Haloarcula marismortui 50S subunit (Hma50) complexed with substrate, intermediate, and product analogues (5)(6)(7)(8)(9). The structures of the Hma50 complexed with a peptidyl-CCA substrate or with an analogue of the intermediate show that the CCA bound in the P-site interacts with the P-loop, nucleotides 2246-2258 of the 23S rRNA, and that the attacking ␣-NH 2 group of a bound CC-puromycin substrate analogue is hydrogen-bonded to both the 2Ј-OH of the terminal A76 of the peptidyl-CCA P-site substrate and to the N3 of the ribosomal base A2451 (Escherichia coli numbering).…”

Teaching argumentation and scientific discourse using the ribosomal peptidyl transferase reaction

“…B: Simple mimics of the peptidyl transferase reaction used to determine the uncatalyzed rate of peptide bond formation [9]. C: Consensus peptidyl transferase reaction where the 2 0 OH of A76 orients the amino group and shuttles protons in the transition state and where a tightly bound water molecule stabilizes the tetrahedral transition state [11]. Figures made using ChemDraw (CambridgeSoft).…”

“…For classroom discussion, three experimental articles were chosen that challenged the original mechanism using the results of different experimental techniques (Table I) [9][10][11]. While reading each article, students are instructed to focus on the main hypothesis, on the connection to the original peptidyl transferase mechanism, and on the proposed changes to the reaction mechanism (Table II).…”

“…[11] Crystal structures with new mimics show that the negatively charged phosphoramide oxygen of CCdAp-Puromycin was directed away from the true oxyanion hole, where now a bound water molecule stabilizes the tetrahedral transition state [11]. The 2 0 OH of the peptidyl-tRNA is also found to have an active role in catalysis where it hydrogen bonds to the nucleophilic amino group and shuttles protons between the amino group and the 3 0 OH of A76 of the peptidyl-tRNA [11].…”

Accommodating the bacterial decoding release factor as an alien protein among the RNAs at the active site of the ribosome

“…This nucleotide has a water molecule positioned close by in structures of the large ribosomal subunit complexed with novel transition state analogues, aimed at unravelling the mechanism of peptide bond formation [75]. The water molecule interacts with the oxyanion of the transition state tetrahedral intermediate.…”