title: RNA-protein binding interface in the telomerase ribonucleoprotein.
abstract: Telomerase is a specialized reverse transcriptase containing an intrinsic telomerase RNA (TR) which provides the template for telomeric DNA synthesis. Distinct from conventional reverse transcriptases, telomerase has evolved a unique TR-binding domain (TRBD) in the catalytic telomerase reverse transcriptase (TERT) protein, integral for ribonucleoprotein assembly. Two structural elements in the vertebrate TR, the pseudoknot and CR4/5, bind TERT independently and are essential for telomerase enzymatic activity. However, the details of the TR-TERT interaction have remained elusive. In this study, we employed a photoaffinity cross-linking approach to map the CR4/5-TRBD RNA-protein binding interface by identifying RNA and protein residues in close proximity. Photoreactive 5-iodouridines were incorporated into the medaka CR4/5 RNA fragment and UV cross-linked to the medaka TRBD protein fragment. The cross-linking RNA residues were identified by alkaline partial hydrolysis and cross-linked protein residues were identified by mass spectrometry. Three CR4/5 RNA residues (U182, U187, and U205) were found cross-linking to TRBD amino acids Tyr503, Phe355, and Trp477, respectively. This CR4/5 binding pocket is distinct and separate from the previously proposed T pocket in the Tetrahymena TRBD. Based on homologous structural models, our cross-linking data position the essential loop L6.1 adjacent to the TERT C-terminal extension domain. We thus propose that stem-loop 6.1 facilitates proper TERT folding by interacting with both TRBD and C-terminal extension. Revealing the telomerase CR4/5-TRBD binding interface with single-residue resolution provides important insights into telomerase ribonucleoprotein architecture and the function of the essential CR4/5 domain.
authors: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA.
authors: Christopher J
authors: CJ
authors: Bley
authors: Xiaodong
authors: X
authors: Qi
authors: Dustin P
authors: DP
authors: Rand
authors: Chad R
authors: CR
authors: Borges
authors: Randall W
authors: RW
authors: Nelson
authors: Julian J-L
authors: JJ
authors: Chen
mesh_terms: D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008957:Models, Genetic; D008958:Models, Molecular; D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000737:chemistry; D012313:RNA / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry*; D012261:Ribonucleoproteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019098:Telomerase / Q000737:chemistry*; D013768:Tetrahymena / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D010455:Peptides; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D012313:RNA; D019098:Telomerase