A mutant of Tetrahymena telomerase reverse transcriptase with increased processivity.

Area of Science:

• Molecular Biology
• Enzymology
• Biochemistry

Background:

• Telomerase protein catalytic subunit (TERT) is a reverse transcriptase (RT) essential for extending chromosomal DNA ends.
• A conserved tyrosine residue precedes catalytic aspartic acids in motif C of retroviral RTs, crucial for catalysis.
• TERTs typically have leucine, valine, or phenylalanine at this position, differing from retroviral RTs.

Purpose of the Study:

• To investigate the functional role of the amino acid preceding motif C in Tetrahymena telomerase.
• To explore the effects of substituting this amino acid with a tyrosine, mimicking retroviral RTs.

Main Methods:

• Development and characterization of a robust in vitro reconstitution system for Tetrahymena telomerase.
• Site-directed mutagenesis to introduce amino acid substitutions at the target position.
• Assays to measure telomerase enzymatic activity, processivity, and DNA primer binding affinity.

Main Results:

• Substitution with tyrosine did not alter overall enzymatic activity but significantly increased processivity.
• Increased processivity correlated with enhanced affinity for the telomeric DNA primer.
• Alanine substitution showed no processivity increase; phenylalanine yielded intermediate effects.

Conclusions:

• The amino acid preceding motif C in TERT plays a role in primer interactions, similar to other RTs.
• Mutating this residue to tyrosine, conserved in retroviral RTs, makes telomerase function more like retroviral RTs.
• Findings provide insights into the structural and functional divergence between telomerase and retroviral reverse transcriptases.