Structural dynamics of the human telomerase ribonucleoprotein complex
Zusammenfassung der Projektergebnisse
The goals as stated in the corresponding research proposal were as follows: 1. Single molecule FRET analysis of hTR and its sub-domains, and the impact of pathogenic mutations; 2. Effect of proteins and their domains on hTR structural dynamics; 3. Structural dynamics of hTR during catalysis. In addition to the experimental work, a considerable amount of time had to be reserved for instrument-specific training, training for programming and data analysis, and adjustments and optimizations of the single-molecule FRET imaging and recording setup. Synthesis of the FRET constructs was achieved relatively fast. After a reasonable amount of optimizations, data of minimal hTR pseudoknot constructs was obtained and could be analyzed. Finally, a construct was obtained that provided a direct measure for pseudoknot formation. Extension of the construct to include additional short structural elements prompted us to investigate the effect of these elements on pseudoknot formation, as well as the effect of a pathogenic mutation. These elements gave new insights into the requirements of pseudoknot formation with respect to buffer composition and sequence requirements. We found that the key structural element in addition to the pseudoknot-forming basepairs is the triplex structure, which consists of several base triplets and is significantly stabilized by Mg2+-ions. Some of the findings were corroborated by NMR experiments performed by the collaborating lab of Prof. Juli Feigon, UC Los Angeles. Finally, we synthesized a construct that was suitable for measuring catalytic telomerase activity of the FRET-labeled RNA. This crucial control experiment was successful, and opened the door for further studies of the telomerase RNP. Due to the availability of a FRET-labeled, catalytically active telomerase RNP complex, we decided to investigate structural dynamics of hTR in this complex. Despite substantial efforts to optimize reconstitution, one major problem remained: As reconstitution of the telomerase RNP is inefficient, single-molecule techniques have to be designed in a way that allows investigation of exclusively catalytically active molecules. Various strategies were followed, and a “proof-of concept” was achieved. However, due to time limitations and problems intrinsic to the telomerase system, only preliminary data was obtained. Various enzymatic and spectroscopic techniques were tested for their suitability to discriminate catalytically active versus inactive molecules of the reconstituted telomerase enzyme.
Projektbezogene Publikationen (Auswahl)
- Single-molecule Analysis of Telomerase Structure and Function. Current Opinion in Chemical Biology, 2011 Dec;15(6):845-52
M. Hengesbach, B.M. Akiyama, and M.D. Stone
- Single-molecule FRET reveals the folding dynamics of the human telomerase RNA pseudoknot domain. Angewandte Chemie International Edition, 2012 Jun 11;51(24):5876-9
M. Hengesbach, N.-K. Kim, J. Feigon, and M.D. Stone
