Probing Na⁺-Induced Changes in the HIV-1 TAR Conformational Dynamics Using NMR Residual Dipolar Couplings:  New Insights into the Role of Counterions and Electrostatic Interactions in Adaptive Recognition

Abstract

Many regulatory RNAs undergo large changes in structure upon recognition of proteins and ligands, but the mechanism by which this occurs remains poorly understood. Using NMR residual dipolar coupling (RDCs), we characterized Na+-induced changes in the structure and dynamics of the bulge-containing HIV-1 transactivation response element (TAR) RNA that mirrors changes induced by small molecules bearing a different number of cationic groups. Increasing the Na+ concentration from 25 to 320 mM led to a continuous reduction in the average inter-helical bend angle (from 46 degrees to 22 degrees ), inter-helical twist angle (from 66 degrees to -18 degrees ), and inter-helix flexibility (as measured by an increase in the internal generalized degree of order from 0.56 to 0.74). Similar conformational changes were observed with Mg2+, indicating that nonspecific electrostatic interactions drive the conformational transition, although results also suggest that Na+ and Mg2+ may associate with TAR in distinct modes. The transition can be rationalized on the basis of a population-weighted average of two ensembles comprising an electrostatically relaxed bent and flexible TAR conformation that is weakly associated with counterions and a globally rigid coaxial conformation that has stronger electrostatic potential and association with counterions. The TAR inter-helical orientations that are stabilized by small molecules fall around the metal-induced conformational pathway, indicating that counterions may help predispose the TAR conformation for target recognition. Our results underscore the intricate sensitivity of RNA conformational dynamics to environmental conditions and demonstrate the ability to detect subtle conformational changes using NMR RDCs.

Thermodynamic examination of trinucleotide bulged RNA in the context of HIV-1 TAR RNA

“…As shown in Figure 3, (Pitt et al 2004;Casiano-Negroni et al 2007). EPR data from Sigurdsson and coworkers showed Na + and Ca 2+ interactions were similar and influence U23; Mg 2+ additionally influences mobility at position U25 (EPR data was not collected for cytosine in the bulge) (Edwards and Sigurdsson 2003).…”

“…The ability of bulged nucleotides to interact with each other and with the neighboring base pairs can effect the local electrostatic environment, hydration, conformations, and ion binding (Auffinger and Westhof 2000;Draper 2004); all of these differences are reflected in the measured DG°3 7 values reported here. In the case of TAR RNA, various conformations have indeed been seen under various ionic conditions (Aboul-ela et al 1995(Aboul-ela et al , 1996Edwards and Sigurdsson 2003;Pitt et al 2004;Casiano-Negroni et al 2007;Zhang et al 2007).…”

“…Transient electric birefringence experiments on TAR RNA show that Mg 2+ ions straighten the bend in TAR RNA by nearly 50% and do not interfere with the binding of arginine (Zacharias and Hagerman 1995a). Residual dipolar coupling NMR experiments show that binding of arginine changes the interhelical angle and arrests interhelical motion; Na + and Mg 2+ stabilize TAR RNA by different modes of interactions (Pitt et al 2004;Casiano-Negroni et al 2007). In EPR studies, Na + and Ca…”

“…Metal ion binding sites are often identified by RNA crystallography when crystals are grown in high concentrations of monovalent and divalent ions (Cate and Doudna 1996;Correll et al 1997;Basu et al 1998). Biophysical studies provide additional data on sites of interaction and binding constants for metal-RNA interactions (Aboul-ela et al 1995;Zacharias and Hagerman 1995a;Edwards and Sigurdsson 2003;Casiano-Negroni et al 2007). However, the extent to which divalent ions stabilize RNA structures, including bulges, is not known.…”

“…The bulge region interacts with the Tat protein, in complex with cyclin T1, to increase the rate of transcription by nearly 100-fold (Coffin et al 1997;Frankel and Young 1998). Hence, TAR RNA has been extensively studied by biochemical and biophysical methods (Muesing et al 1987;Dingwall et al 1989;Cordingley et al 1990;Weeks et al 1990;Calnan et al 1991;Puglisi et al 1992;Tao and Frankel 1992;Aboul-ela et al 1995Aboul-ela et al , 1996Zacharias and Hagerman 1995a;Ippolito and Steitz 1998;Edwards and Sigurdsson 2003;Casiano-Negroni et al 2007). The trinucleotide (UCU) bulge is at position 23-25 (HIV-1 TAR RNA numbering system is being used here) (Fig.…”