Hydrogen-Deuterium Exchange of Tetrahymena Group I Ribozyme

Abstract

Tetrahymena group I intron has served as good model system for conformation or dynamics study of large RNA. The Tetrahymena L-21 ScaI ribozyme is derived from the self-splicing group I intron. The internal guide sequence (IGS) in the ribozyme forms the P1 duplex with a substrate and then specific site of substrate was cleaved by attack of an exogenous guanosine. X-ray crystal structures of group I intron from several species have been solved and useful in the understanding of previous biochemical and biophysical study. The dynamics study of ribozyme is motivated by the desire to understand the conformational states and flexibility of ribozyme relating RNA function. The measurement of hydrogen exchange rates has been an important tool for studying the macromolecular conformation or dynamics. The imino proton resonances of nucleic acids are good probes of the hydrogen exchange study for studying the dynamics of each base pair. The exchange rates of imino protons depend on not only the base pair stability but also solvent accessibility. Hydrogen-deuterium (H/D) exchange method is widely used to measure the exchange rates of slowly exchanging imino protons whereas the exchange rates of fast-exchanging imino protons can be determined by water magnetization transfer method of NMR. It is very difficult to study the dynamics or conformational change of the Tetrahymena ribozyme by using NMR spectroscopy because whole ribozyme is too large (greater than 120 kDa). The NMR study on the dynamics or flexibility of the ribozyme can provide lots of information about the long-range molecular interaction, tertiary folding, or enzymatic kinetics in the large-sized ribozyme. The problem caused from resonance assignment may be solved by incorporation of the site-specific isotope-labeling method. However, it is still unclear which NMR method successfully provides the answer for the question on the dynamics or structure of the 120-kDa ribozyme. Here, we first challenged the H/D exchange study of 120-kDa ribozyme by NMR in order to investigate the dynamics property of very large RNA molecule. The success of this experiment suggests that the Tetrahymena ribozyme can be studied on its dynamics or conformational change in the molecular level when the sitespecific isotope-labeled ribozyme is prepared. The purified Tetrahymena group I ribozyme was kindly gifted by Prof. Daniel Herschlag (Stanford University). Ribozyme was buffer exchanged into 10 mM Mg NMR buffer (10 mM sodium phosphate pH 6.6, 100 mM NaCl, 10 mM MgCl2, 0.1 mM EDTA in 90% H2O/10% D2O). All NMR experiments were performed on Varian Inova 500 or 600 MHz spectrometer equipped a triple resonance or cold probe. The NMR data were processed using FELIX2004 (Accelrys) as described previously. 1D proton spectra were collected using a gradient 11 echo pulse sequence for water suppression. The ribozyme sample in 90% H2O/10% D2O NMR buffer was exchanged into100% D2O NMR buffer by using a 1mL bed volume G-25 size exclusion spin column. 1D imino proton spectra were acquired as function of time after deuterium exchange. The intensities of some slowly exchanging imino proton resonances were fit to the simple exponential decay I(t) = I0e −t/τex + c in Sigma plot, where I(t) and I0 are peak intensities at time t and zero, respectively, and τex is the exchange time. 9