Discrimination between transfer-RNAs by tyrosyl-tRNA synthetase

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Tyrosyl-tRNA synthetase (TyrRS) is crucial for protein synthesis, attaching tyrosine to its cognate tRNA(Tyr).
• Understanding synthetase-tRNA interactions is vital for deciphering the fidelity of the genetic code.

Purpose of the Study:

• To construct and validate a molecular model of the Bacillus stearothermophilus TyrRS:tRNA(Tyr) complex.
• To elucidate the specific molecular determinants governing TyrRS recognition and discrimination of tRNA(Tyr).

Main Methods:

• Iterative cycles of molecular modeling, site-directed mutagenesis of TyrRS, and biochemical assays.
• Comparison with crystal structures of related synthetase-tRNA complexes.
• Development of an in vivo system to assess tRNA discrimination by synthetase mutants.

Main Results:

• The model indicates TyrRS approaches tRNA(Tyr) at the variable loop, with the anticodon being the primary identity element.
• Recognition of the discriminator base A73 involves specific chemical groups on TyrRS, optimized in the transition state.
• Mutagenesis identified residue Glu152 as a negative determinant, preventing binding of non-cognate tRNAs via repulsion.

Conclusions:

• The anticodon and discriminator base are critical for TyrRS:tRNA(Tyr) recognition.
• Specific amino acid residues, like Glu152, play key roles in ensuring synthetase fidelity by repelling incorrect tRNAs.
• Evolution has conserved local sequence and structure in TyrRS for efficient tRNA recognition and charging.