High-resolution NMR Studies on tRNA Structure in Solution

Abstract

The advent of the yeast tRNA^phe crystal structure (Kim et al. 1974; Robertus et al. 1974) stimulated a variety of biophysical studies on the conformations and dynamics of tRNA in solution. Proton nuclear magnetic resonance (NMR) spectroscopy has emerged as one of the most important approaches to this problem. The low-field (−15 to −11 ppm) region of the proton NMR spectrum of most tRNAs contains 20 secondary-structure and 7 ± 1 tertiary-structure base-pair resonances (the hydrogen-bonded ring nitrogen proton of U [UN3H] or G [GN1H] of base pairs with hydrogen-bond lifetimes of 5 msec or greater). Once assigned, these resonances provide the basis by which the conformation and dynamics of individual base pairs in the tRNA molecule can be monitored. The first priority of this type of research is reliable assignments; without these, studies on conformational changes, helix-coil dynamics, etc., are either useless or misleading. The first part of this paper deals with assignment and resolution. Poor spectral resolution limits the precision of NMR analysis and interpretation. A second type of resolution, namely, time resolution, will be discussed in the second part of this paper.

OBTAINING NMR SPECTRA
The low-field NMR spectra that we present were obtained at 360 MHz on a modified Bruker HXS360 spectrometer at the Stanford Magnetic Resonance Laboratory. Typically, a 6-mg sample of a pure tRNA was dissolved in 0.18ml of appropriate buffer and transferred to a Wilmad 508CP NMR microtube. Spectra were obtained using either correlation spectroscopy (Dadok and Sprecher 1974) with a 2500-Hz/sec sweep...