A chimaeric glutamyl:glutaminyl-tRNA synthetase: implications for evolution.

Area of Science:

• Molecular Biology
• Protein Evolution
• Enzymology

Background:

• Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that catalyze aminoacylation of tRNA.
• aaRSs have evolved through domain acquisition, with the anticodon binding domain added to the catalytic domain.
• Bacterial glutamyl-tRNA synthetase (GluRS) and glutaminyl-tRNA synthetase (GlnRS) anticodon binding domains are structurally distinct, reflecting unique evolutionary histories.

Purpose of the Study:

• To investigate the properties of a putative ancestral eukaryotic GluRS formed after anticodon binding domain acquisition.
• To construct and characterize a chimeric protein (cGluGlnRS) combining the catalytic domain of E. coli GluRS and the anticodon binding domain of E. coli GlnRS.

Main Methods:

• Construction of a chimeric protein, cGluGlnRS.
• Assays for glutamylation activity of E. coli tRNA(glu).
• Complementation studies using a temperature-sensitive E. coli GluRS mutant.
• Determination of tRNA binding affinity.
• Comparative analysis of anticodon binding domain structures.

Main Results:

• The chimeric cGluGlnRS exhibited detectable glutamylation activity and could complement a temperature-sensitive E. coli GluRS strain.
• cGluGlnRS and native E. coli GluRS bound E. coli tRNA(glu) with similar affinity, indicating the anticodon binding domain enhances catalytic efficiency (kcat).
• An extended loop in GlnRS anticodon binding domains, absent in archaeal GluRS, suggests a later evolutionary addition for enhanced discrimination.

Conclusions:

• The anticodon binding domain significantly enhances the catalytic activity of GluRS for glutamylation.
• The chimeric protein provides insights into the early evolution of eukaryotic GluRS after anticodon binding domain acquisition.
• Structural differences in anticodon binding domains highlight the distinct evolutionary paths of bacterial GluRS and GlnRS.