Magnesium Dependence of the Amplified Conformational Switch in the Trans-Acting Hepatitis Delta Virus Ribozyme

Abstract

The ability of divalent metal ions to participate in both structure formation and catalytic chemistry of RNA enzymes (ribozymes) has made it difficult to separate their cause and effect in ribozyme function. For example, the recently solved crystal structures of precursor and product forms of the cis-cleaving genomic hepatitis delta virus (HDV) ribozyme show a divalent metal ion bound in the active site that is released upon catalysis due to an RNA conformational change. This conformational switch is associated with a repositioning of the catalytically involved base C75 in the active-site cleft, thus controlling catalysis. These findings confirm previous data from fluorescence resonance energy transfer (FRET) on a trans-acting form of the HDV ribozyme that found a global conformational change to accompany catalysis. Here, we further test the conformational switch model by measuring the Mg(2+) dependence of the global conformational change of the trans-acting HDV ribozyme, using circular dichroism and time-resolved FRET as complementary probes of secondary and tertiary structure formation, respectively. We observe significant differences in both structure and Mg(2+) affinity of the precursor and product forms, in the presence and absence of 300 mM Na(+) background. The precursor shortens while the product extends with increasing Mg(2+) concentration, essentially amplifying the structural differences observed in the crystal structures. In addition, the precursor has an approximately 2-fold and approximately 13-fold lower Mg(2+) affinity than the product in secondary and tertiary structure formation, respectively. We also have compared the C75 wild-type with the catalytically inactive C75U mutant and find significant differences in global structure and Mg(2+) affinity for both their precursor and product forms. Significantly, the Mg(2+) affinity of the C75 wild-type is 1.7-2.1-fold lower than that of the C75U mutant, in accord with the notion that C75 is essential for a catalytic conformational change that leads to a decrease in the local divalent metal ion affinity and release of a catalytic metal. Thus, a consistent picture emerges in which divalent metal ions and RNA functional groups are intimately intertwined in affecting structural dynamics and catalysis in the HDV ribozyme.

Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape

“…Taken together, these observations strongly support the result from our EMSAs that the precursor and product forms of the most recently crystallized trans-acting ribozyme version display distinct global conformations. Thus, a global conformational change appears to accompany cleavage, consistent with previous findings on other transacting HDV ribozymes Pereira et al 2002;Jeong et al 2003;Tinsley et al 2004).…”

“…In addition to numerous studies of self-cleavage by the cisacting genomic and antigenomic HDV ribozymes (Been and Wickham 1997;Perrotta et al 1999aPerrotta et al ,b, 2006Wadkins et al 1999;Nakano et al 2000Nakano et al , 2003Shih and Been 2002;Harris et al 2004;Been 2006;Chadalavada et al 2007;Gong et al 2007Gong et al , 2009Sefcikova et al 2007a,b;Cerrone-Szakal et al 2008;Gong et al 2008;Chen et al 2009), significant work has also been performed to investigate trans-acting forms of this ribozyme for enzymology and therapeutic purposes (Been 1994;Luptak et al 2001;Harris et al 2002;Pereira et al 2002;Jeong et al 2003;Tinsley et al 2003Tinsley et al , 2004Das and Piccirilli 2005;Tinsley and Walter 2007;Walter and Perumal 2009). Generally, trans-acting HDV ribozymes exhibit a 1-2 order-of-magnitude slower catalytic rate constant compared to their cis-acting counterparts, likely due to a less tightly knit structure (Tinsley and Walter 2007).…”

Evidence that binding of C5 protein to P RNA enhances ribozyme catalysis by influencing active site metal ion affinity

“…Both classes can make significant contributions to function that are reflected in Mg 2+ titration experiments, which are difficult to interpret in terms of the thermodynamics of individual metal ion interactions. Additionally, it is well established in other ribozyme systems that Mg 2+ binding can increase catalytic rate constants indirectly by stabilizing active RNA conformations or by promoting substrate docking steps (e.g., Shan and Herschlag 2002;Tinsley et al 2004). Thus, while the contribution of the protein subunit to P RNA catalysis appears to be related to the binding of Mg 2+ ions, the binding sites and functional roles of these ions are fundamentally difficult to establish.…”

HDV Family of Self-Cleaving Ribozymes

“…In solution, however, a FRET-based assay measured a significant change in the overall shape 134 Nathan Riccitelli and Andrej Lupták of the molecule before and after the reaction. 77,86,87 When the ribozyme is labeled at the top of P2 and bottom of P4 helices, an inhibited precursor ribozyme showed compaction upon Mg 2þ binding, whereas the product form showed extension of the molecule (approximately 50 and approximately 66 Å , respectively). These results suggest that the presence of the leader sequence in the precursor ribozyme causes bending in the structure such that the distal ends of P2 and P4 are closer before the reaction and further apart in the product form.…”

“…Mg 2þ -dependent conformational changes differed for the C75 and C75U variants, where the wild-type ribozyme showed shorter P2-P4 end-to-end distance in both precursor and product states. 87 …”