Substrate-mediated fidelity mechanism ensures accurate decoding of proline codons

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Aminoacyl-tRNA synthetases (aaRS) are crucial for protein synthesis, attaching specific amino acids to cognate tRNAs.
• Prolyl-tRNA synthetases (ProRS) can mischarge tRNA(Pro) with alanine or cysteine.
• Quality control mechanisms, including the INS editing domain and YbaK, ensure proline codon accuracy.

Purpose of the Study:

• To investigate the catalytic mechanism and substrate recognition of the freestanding editing domain homolog, YbaK from *Hemophilus influenzae*.
• To elucidate how YbaK specifically deacylates Cys-tRNA(Pro) without affecting Pro-tRNA(Pro).

Main Methods:

• Site-directed mutagenesis to identify key catalytic residues in YbaK.
• Enzymatic assays using isosteric substrates and functional group analogs.
• Computational modeling to understand substrate binding and transition states.
• Mass spectrometry to characterize reaction products.

Main Results:

• YbaK utilizes a novel substrate-assisted catalytic mechanism for Cys-tRNA(Pro) deacylation.
• This mechanism involves thiol-specific chemistry, distinguishing it from the INS domain's steric exclusion.
• YbaK effectively prevents the hydrolysis of correctly charged Pro-tRNA(Pro).

Conclusions:

• The INS and YbaK domains evolved distinct strategies to ensure accurate proline decoding.
• YbaK's thiol-specific chemistry provides a complementary quality control mechanism to the INS domain's steric exclusion.
• These findings highlight the intricate molecular mechanisms maintaining translational fidelity.