Transfer RNA-mediated editing in threonyl-tRNA synthetase. The class II solution to the double discrimination problem

Area of Science:

• Molecular Biology
• Enzymology
• Structural Biology

Background:

• Threonyl-tRNA synthetase (ThrRS) is a class II synthetase crucial for protein synthesis.
• Accurate aminoacylation of tRNA is vital, requiring discrimination against similar amino acids like valine.
• Previous studies indicated ThrRS uses a unique zinc ion for discrimination.

Purpose of the Study:

• To investigate the discrimination mechanism of ThrRS against non-cognate amino acids, specifically serine.
• To elucidate the role of the N-terminal domain in the editing of mischarged tRNA(Thr).
• To compare the editing mechanisms of class I and class II synthetases.

Main Methods:

• Crystal structure analysis of ThrRS with a seryl adenylate analog.
• Biochemical assays to study aminoacylation and hydrolysis.
• Comparative analysis of tRNA editing mechanisms.

Main Results:

• The crystal structure revealed that serine cannot be fully discriminated during the activation step.
• Hydrolysis of the mischarged ser-tRNA(Thr) occurs at a distinct site within the N-terminal domain of ThrRS.
• Both classes of synthetases utilize tRNA CCA end conformational switching for aminoacylation and editing.

Conclusions:

• ThrRS employs a specific N-terminal editing site for hydrolyzing mischarged ser-tRNA(Thr).
• The editing mechanisms of class I and class II synthetases are proposed as mirror images, consistent with tRNA binding and amino acid activation.
• Conserved tRNA CCA end conformational flexibility underpins accurate aminoacylation and editing across synthetase classes.