Divergent Evolution of Eukaryotic CC- and A-Adding Enzymes

Area of Science:

• Molecular Biology
• Evolutionary Biology
• Biochemistry

Background:

• Transfer RNA (tRNA) CCA end synthesis is crucial for cellular function.
• This synthesis is typically performed by CCA-adding enzymes or through collaborative partial activities.
• Partial CCA-adding activities were previously thought to be exclusive to bacteria.

Purpose of the Study:

• To investigate the presence and evolutionary origins of CC- and A-adding enzymes in the choanoflagellate *Salpingoeca rosetta*.
• To compare the evolutionary pathways and molecular mechanisms of partial CCA-adding activities in eukaryotes and bacteria.
• To elucidate the role of specific molecular elements in the evolution of tRNA nucleotidyltransferases.

Main Methods:

• Bioinformatic analysis to identify CC- and A-adding enzymes in *Salpingoeca rosetta*.
• Comparative analysis of enzyme sequences and evolutionary relationships.
• Site-directed mutagenesis to investigate the functional role of conserved loop regions.

Main Results:

• Identified distinct CC- and A-adding enzymes in *Salpingoeca rosetta* with separate evolutionary origins.
• Demonstrated that eukaryotic CC-adding enzymes evolved differently from bacterial counterparts but share similar molecular constraints.
• Restored full CCA-adding activity in eukaryotic enzymes by introducing specific loop elements, highlighting conserved mechanistic principles.

Conclusions:

• Partial CC- and A-adding activities in eukaryotes and bacteria are mechanistically similar but evolved independently.
• Specific conserved positions in a flexible loop region are critical for the catalytic activity of tRNA nucleotidyltransferases.
• This study expands the understanding of tRNA modification evolution across different domains of life.