Two purified domains of telomerase reverse transcriptase reconstitute sequence-specific interactions with RNA

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Telomerase is a ribonucleoprotein enzyme essential for maintaining telomere length.
• Telomerase reverse transcriptase (TERT) and telomerase RNA (TER) are core components of telomerase.
• Understanding the interaction between TERT and TER is key to elucidating telomerase assembly and function.

Purpose of the Study:

• To identify and characterize the domains of Tetrahymena TERT that directly interact with TER.
• To define the specific sequence and structural requirements of TER for recognition by TERT RNA-binding domains.
• To investigate the relationship between TER binding sites and the catalytic activity of telomerase.

Main Methods:

• Bacterial expression of Tetrahymena TERT domains.
• Quantitative binding assays to measure TERT-TER interactions.
• Analysis of TER sequence and structure requirements for TERT binding.

Main Results:

• Identified specific TERT domains that bind TER independently of assembly chaperones.
• Defined distinct, non-overlapping TER sequence and structural requirements for two TERT RNA interaction domains.
• Demonstrated that key TER residues for TERT binding are a subset of those required for catalytic activity.

Conclusions:

• Telomerase exhibits functional specialization through an intricate ribonucleoprotein architecture.
• The identified TERT domains and their TER binding sites are separable from the catalytic active site.
• These findings provide insights into the structural basis of telomerase assembly and regulation.