RNA-protein binding interface in the telomerase ribonucleoprotein.

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Telomerase is a reverse transcriptase essential for telomere maintenance.
• It comprises telomerase RNA (TR) and telomerase reverse transcriptase (TERT) protein.
• The TR-binding domain (TRBD) of TERT is crucial for assembling the telomerase ribonucleoprotein complex.

Purpose of the Study:

• To elucidate the molecular details of the interaction between the CR4/5 domain of TR and the TRBD of TERT.
• To map the RNA-protein binding interface at single-residue resolution.

Main Methods:

• Photoaffinity cross-linking using photoreactive 5-iodouridines incorporated into medaka CR4/5 RNA.
• UV cross-linking of RNA to medaka TRBD protein.
• Identification of cross-linked RNA and protein residues via alkaline hydrolysis and mass spectrometry.

Main Results:

• Identified three CR4/5 RNA residues (U182, U187, U205) cross-linking to specific TRBD amino acids (Tyr503, Phe355, Trp477).
• The CR4/5-TRBD binding pocket is distinct from previously characterized TERT binding sites.
• Cross-linking data suggest loop L6.1 is positioned near the TERT C-terminal extension domain.

Conclusions:

• The CR4/5-TRBD interaction is critical for telomerase ribonucleoprotein architecture.
• Loop L6.1 may facilitate TERT folding through interactions with TRBD and the C-terminal extension.
• This detailed mapping provides insights into telomerase assembly and function.