RNA Dynamics by Solution and Solid State Deuterium NMR

University Of Washington, Seattle WA

Investigators

Abstract

The objective of this project is to investigate nucleic acids dynamics by applying solution and solid-state NMR conjointly to a paradigmatic system, the HIV-1 TAR. RNA dynamics will be described more comprehensively than it has been possible so far by analyzing the experimental results obtained by the two NMR methods through a common theoretical framework. The concomitant use of the two spectroscopies will allow a wider range of time scales and sites than would be possible by using either technique in isolation, and to compare dynamics of RNA in solution and the solid. The central hypothesis underlying this project is that motion is an important aspect of nucleic acid function and HIV TAR provides an eloquent demonstration of this premise. Its structure and molecular interactions have been studied extensively, yet the molecular basis for TAR recognition by Tat protein is poorly understood because of complex conformational dynamics in the protein and RNA. Both solution and solid state NMR are powerful methods to study dynamics, but each has limitations. Solution relaxation data are poorly sensitive to motions that occur at certain rates, while solid-state deuterium lineshape analysis requires singly deuterated samples. Therefore, it is necessary to develop new methods to collect and analyze the experimental results to understand in quantitative detail how motion contributes to molecular recognition in nucleic acids. By analyzing the two set of spectroscopic results through the same theoretical framework, it will be possible to gain qualitatively and quantitatively new insight into the nature of the motions within TAR RNA that underlie its recognition by Tat protein and therefore its function in gene regulation. The new tools to study functional dynamics in nucleic acids developed in this project will be broadly applicable to other nucleic acid and even to proteins. This project is highly interdisciplinary, crossing the areas of physical chemistry, molecular biology, biochemistry, molecular pharmacology and molecular virology. Therefore, it provides many opportunities for scientific collaborations and for exposing undergraduate and graduate students to interdisciplinary research. It provides fertile grounds for training future leaders in the development and application of NMR methods to study biological systems. An important aspect of the PI's research group is the presence of a majority of female students and a number of under-represented minorities. One of the PIs supervises every summer a high school student from an under-represented minority group through the University of Washington Genome and STAR programs aimed at increasing recruitment of under-represented minority students to the science