Spectroscopic and thermodynamic studies of conformational changes in long, natural messenger ribonucleic acid molecules

Abstract

Physical techniques have proved to be very useful in the analysis of biological molecules. In this communication, we report the first circular dichroism (CD) and optical melting studies, coupled with thermodynamic estimations, on conformational changes in two naturally occurring messenger RNAs. One of the mRNAs analysed contained a very stable stem-loop structure (termed REP sequences), and the other was identical in sequence, except for a deletion of the stem-loop. The CD spectra of both RNAs show that they are in the A-conformation and are extensively base-paired and stacked even at low ionic strength. Optical melting studies show that the RNA with out the stem-loop structure melts in a single co-operative transition with a Tm of 65.5 °C whereas the RNA containing the stem-loop structure has three structural transitions with Tm values of 66.5, 74.5 and 86.5 °C. These data are consistent with those obtained using CD and together they show that the stem-loop structure melts in two distinct stages. Addition of spermidine increases the temperature at which these transitions occur and alters the conformation of the stem loop in such a way that it melts in one rather than two transitions. We present a method for calculating ΔH° and ΔS° of each transition and show that the thermodynamic values estimated are consistent with the observed results. Our results demonstrate that CD and optical melting spectroscopy, together with thermodynamic calculations, can be successfully used to gain insights into the dynamic structural features of complex RNAs.