Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi‡

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Telomerase, comprising telomerase reverse transcriptase (TERT) and template RNA (TER), is vital for cellular processes like aging, cancer, and stem cell renewal.
• Conserved structural elements in TER, including the template-pseudoknot (T-PK) and helical three-way junction (TWJ), are essential for telomerase function.
• Understanding the species-specific structural and functional nuances of telomerase is critical for elucidating its molecular mechanisms.

Purpose of the Study:

• To investigate the structural and functional characteristics of N-terminally truncated TERTs from Candida albicans and Candida tropicalis.
• To explore the role of the template-pseudoknot (T-PK) and helical three-way junction (TWJ) in fungal TERT activity.
• To identify novel structural motifs that regulate telomerase catalytic function.

Main Methods:

• X-ray crystallography and cryo-electron microscopy were employed to determine the structures of Candida TERTs.
• Biochemical assays were performed to assess the reverse transcriptase activity of TERT proteins in various conformations and complexes.
• Structural analysis was conducted to identify conserved motifs and their interactions within the TERT structure.

Main Results:

• Candida TERT proteins exhibited a single round of telomere addition, irrespective of T-PK/TWJ presence, and displayed standard reverse transcriptase activity.
• The C-terminal domain of TERT was found to exist in at least two distinct conformations, with interconversion regulating catalytic activity.
• A conserved tertiary structural motif, the U-motif, was identified; it interacts with the reverse transcriptase domain and is essential for catalytic activity.

Conclusions:

• Fungal TERTs share common characteristics with other TERTs but also possess unique species-specific features.
• The identified U-motif is a key determinant of fungal TERT catalytic activity, highlighting its importance in telomerase mechanics.
• These findings provide novel insights into the structure, conformational dynamics, and catalytic regulation of fungal telomerase.