Distance measurements in RNA using DEER spectroscopy with site-directed spin labeling
EMSL Project ID
3151
Abstract
The goal of this work is to investigate folding and function in structured RNA molecules through distance measurements using pulsed EPR methodologies in conjunction with site-specific spin labeling techniques. In initial studies, model systems consisting of a series of well-characterized RNA duplexes containing covalently linked nitroxide spin labels will be examined in order to obtain an ?RNA distance ruler?. Distance measurements will be performed using the pulsed EPR technique of double electron-electron- resonance (DEER) spectroscopy, which allows determination of interspin distances > 30 ?. Measured distances will be compared with predictions based on molecular modeling. DEER measurements will subsequently be applied to three different structured RNA molecules whose folding pathways are predicted to be highly Mg2+-ion dependent. These folding pathways can be tested by placing spin labels at strategic locations in the RNA and measuring interspin distances as a function of added Mg2+. RNA systems that will be studied include two different sequences of the Hammerhead Ribozyme and one construct of the P5abc subdomain of the Group I intron. Collaborators on this project include a graduate student and faculty member at Texas A&M University and Dr. Michael Bowman of PNLL, who has demonstrated feasibility of DEER measurements using model systems. This work should establish the utility of pulsed EPR methods for determining structure and rearrangement in complex RNA systems.
Project Details
Project type
Capability Research
Start Date
2002-12-02
End Date
2005-12-04
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members