Structure of Human Telomerase RNA
EMSL Project ID
2574
Abstract
Significance - Telomerase is a ribonucleoprotein responsible for the replication of the chromosome termini. It is composed of a RNA of 400 nucleotides and several proteins that carry out the enzymatic activity of the holoenzyme and promote its stability. The RNA also performs essential functions in regulating the unusual processivity profile of telomerase and other properties of this enzyme. Since telomerase is activated in most human cancers, its inhibition could provide new antineoplastic agents with broad activity. Despite its importance, there are at the moment no high-resolution structures for any of the protein components of telomerase nor any of the essential structural domains of its RNA . This is a critical limiting point in efforts directed at addressing the biological function of this enzyme and in devising new ways to inhibit its function in transformed cells. Obtaining the NMR structure of critical domains of telomerase RNA would allow deeper insight into its biological function and the design of new inhibitors that regulate its activity. We are studying structure/function relationships of two domains of human telomerase RNA (hTR) responsible for enzyme activation and RNP assembly and of the proteins that interact with these domains. Objective - We are studying the structure of the so-called activation domain of hTR. This is a region of 60 nucleotides that activates catalysis by the reverse transcriptase enzyme component of telomerase. Our aim is to dissect its structure, conformational flexibility and structure-function relationship through a combination of NMR structure determination, including new methods based on residual dipolar couplings. In the long term, we aim to use this information for drug design, and the quality of the structure is of the utmost importance. We are determining by NMR the structures of two highly sequences within this region that, when mutated, abrogate telomerase activity in vitro and in vivo. A first structure is nearing completion, and only requires dataset at 800 Mhz in order to obtain the information needed to complete the structure. Concerning the second essential telomerase structural domain, we have conducted advanced NMR studies of the apical part of the domain that is absolutely necessary to enzymatic activity: mutations in this region are just as deleterious as mutating key catalytic residues within the enzymatic protein component. The NMR data we have obtained so far are of excellent quality (See attached file) and it is clear that high-resolution structure determination by NMR will be possible. We seek access at PNNL in order to collect data that are required to complete the dataset needed for successful structure determination Instrumentation required - 1. 6 days of time at 800 Mhz in order to collect NOESY build-up data to complete the NOE-based dataset needed for structure calculations. Because of the crowdiness of the NMR spectra of RNA, it is essential that such data are collected at the highest possible field in order for the greatest number of NOEs to be collected and interpreted. The University of Washington has a 750 Mhz spectrometer, the quality of the data is at the moment insufficient: the facility is being upgraded by the purchase of a new probe to increase sensitivity and by the stabilization of the temperature of the room to improve long-term instrumental stability. 2. 9 days of time on a 600 Mhz spectrometer in order to collect 3-dimensional NOESY spectra (13C-edited) of a 13C-labeled NMR sample of the same RNA at different mixing time, as well as a 13C-edited TOCSY spectrum. These data are required to complete the data set of NOE-based distance restraints and scalar coupling constants required to determine RNA structure of sufficient high quality. In Seattle, we do not have a 600 mhz spectrometer; the increased sensitivity and resolution of 600 Mhz data provides very significant advantages.
Project Details
Project type
Capability Research
Start Date
2002-10-01
End Date
2003-04-24
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members