Participation of the tRNA A76 hydroxyl groups throughout translation

Abstract

The free 2'-3' cis-diol at the 3'-terminus of tRNA provides a unique juxtaposition of functional groups that play critical roles during protein synthesis. The translation process involves universally conserved chemistry at almost every stage of this multistep procedure, and the 2'- and 3'-OHs are in the immediate vicinity of chemistry at each step. The cis-diol contribution affects steps ranging from tRNA aminoacylation to peptide bond formation. The contributions have been studied in assays related to translation over a period that spans at least three decades. In this review, we follow the 2'- and 3'-OHs through the steps of translation and examine the involvement of these critical functional groups.

1. Base-catalyzed isomerization of aminoacyl tRNAs

When the vicinal hydroxyl of an aminoacyl tRNA nucleophilically attacks the neighboring ester linkage, the amino acid can be transferred to the attacking hydroxyl.

2. X-ray crystal structure of E. coli tRNA^Asp and aspartyl-adenylate bound in the active site of aspartyl tRNA synthetase [PDB entry 1C0A(26)]

The A76 3'-OH is positioned for attack on the aspartyl-adenylate carboxyl carbon. The 2'-OH hydrogen bonds with serine 193, which is invariant in all AspRSs, and the aspartyladenylate α-amino group. These interactions contribute to the organization of the active site prior to aminoacylation.

3. X-ray crystal structure of the post-transfer editing substrate analog 2_-(L-norvalyl)amino-2_-deoxyadenosine bound to the editing site of Thermus thermophilus Leucyl tRNA synthetase [PDB entry 1OBC(45)]

The A76 3'-OH is positioned to hydrogen bond with the _-OHs of threonines 247 and 248, and the backbone amide of threonine 248. The 3'-OH also coordinates a specifically bound water molecule that may be directly involved in hydrolysis of the editing substrate.

4. X-ray crystal structure of Saccharomyces cerevisiae Phe-tRNA^Phe bound to Thermus aquaticus EF-Tu [PDB entry 1TTT(55)]

Numerous hydrogen bonds between EF-Tu and the aminoacyl tRNA stabilize the 3' configuration.

5. The P-site A76 2'-OH is positioned for a critical role in proton transfer

(A) X-ray crystal structure of ribosomal substrates bound in the active site [PDB entry 1VQN (78)]. The α-NH₃ group of the A-site substrate has been replaced with a hydroxyl group to inhibit the reaction. The A76 2'-OH hydrogen bonds with the α-NH₃, and the 2'- and 3'-OHs coordinate a solvent molecule on their far side. (B) Schematic of the peptidyl transfer intermediate and the proposed mechanism for resolution into products. The 2'-OH acts as a proton shuttle by simultaneously donating a proton to the 3'-OH and accepting a proton from the α-NH₃. (C) X-ray crystal structure of the transition state mimic CCA-phosphate-puromycin_Phe-CC [PDB entry 1VQP (78)]. The non-bridging phosphate oxygens mimic the peptide and the oxyanion. The proposed proton transfers are indicated with arrows.