Evolution of tRNA nucleotidyltransferases: a small deletion generated CC-adding enzymes

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• CCA-adding enzymes synthesize the 3' CCA terminus of tRNA without a template.
• Some organisms utilize related enzymes with partial CC or A addition activities.
• The catalytic mechanisms and evolutionary origins of these partial activities remain unclear.

Purpose of the Study:

• Investigate the molecular basis for the restricted activity of CC-adding enzymes.
• Identify the specific protein regions responsible for full CCA-adding function.
• Elucidate the evolutionary relationship between CCA-, CC-, and A-adding enzymes.

Main Methods:

• Recombinant protein expression and activity assays.
• Site-directed mutagenesis and chimera construction.
• Phylogenetic analysis of tRNA nucleotidyltransferases.

Main Results:

• A novel region outside conserved motifs was identified, crucial for A-addition.
• Introducing this region into CC-adding enzymes restored full CCA-adding activity.
• Deletion of this region in CCA-adding enzymes resulted in CC-adding activity, identifying a hallmark for CC-adding enzymes.
• Phylogenetic analysis suggests CC-adding enzymes evolved multiple times from CCA-adding enzymes.

Conclusions:

• A specific protein region dictates the A-incorporating activity of CCA-adding enzymes.
• The evolution of CC-adding enzymes from CCA-adding enzymes is characterized by the loss of this region.
• This region's presence or absence serves as a key indicator for enzyme activity and evolutionary history.