A single-molecule assay for telomerase structure-function analysis.

Nucleic Acids Research
John Y WuXiaowei Zhuang

Abstract

The activity of the telomerase ribonucleoprotein enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report a single-molecule assay for direct structural analysis of catalytically active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity, allowing precise discrimination between inactive, active and processive enzyme binding events. FRET signals from enzyme molecules during the active and processive binding events were then used to determine the global organization of telomerase RNA within catalytically active holoenzymes. Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures.

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Methods Mentioned

BETA
footprinting
FRET
PCR
Electrophoresis
dot blot
chip

Software Mentioned

LabView
IDL
ImageQuant

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