Role of a Heterogeneous Free State in the Formation of a Specific RNA−Theophylline Complex

Abstract

The helical regions of RNA are generally very stable, but the single-stranded and loop regions often exist as an ensemble of conformations in solution. The theophylline-binding RNA aptamer forms a very stable structure when bound to the bronchodilator theophylline, but the theophylline binding site is not stably formed in the absence of ligand. The kinetics for theophylline binding were measured here by stopped-flow fluorescence spectroscopy to probe the mechanism for theophylline binding in this RNA aptamer. The kinetic studies showed that formation of the RNA-theophylline complex is over 1000 times slower than a diffusion-controlled rate, and the high affinity of the RNA-theophylline complex arises primarily from a slow dissociation rate for the complex. A theophylline-independent rate was observed for formation of the theophylline-RNA complex at high theophylline concentration, indicating that a conformational change in the RNA is the rate-limiting step in complex formation under these conditions. The RNA-theophylline complex requires divalent metal ions, such as Mg2+, to form a high-affinity complex, and there is a greater than 10000-fold reduction in affinity for theophylline in the absence of Mg2+. This decrease in binding affinity in the absence of Mg2+ results primarily from an increased dissociation rate for the complex. The implications of an ensemble of conformations in the free state of this theophylline-binding RNA are discussed and compared with mechanisms for formation of protein-ligand complexes.

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

“…Within these energy landscapes are "off-pathway" species that form nonnative interactions and require additional rearrangements to attain functional states. Indeed, conformational transitions have been inferred within the active sites of ribozymes (e.g., Chanfreau and Jacquier 1996;Wang et al 1999;Zhuang et al 2002a;Schmeing et al 2005;Hougland et al 2006;Martick and Scott 2006;Hsieh and Fierke 2009;Marcia and Pyle 2012;Sripathi et al 2014) and in the binding sites of in vitro-selected aptamers (e.g., Jucker et al 2003;Flinders et al 2004;DuchardtFerner et al 2010), perhaps reflecting RNA's difficulty in specifying a unique structure. Nature appears to have harnessed this feature to regulate gene expression via conformational transitions of riboswitches (e.g., Wickiser et al 2005;Gilbert et al 2006;Noeske et al 2007;Ottink et al 2007;Montange and Batey 2008) and coordinate complex multistep processes such as pre-mRNA splicing and protein synthesis (Staley and Guthrie 1998;Guo et al 2009;Frank and Gonzalez 2010;Voorhees and Ramakrishnan 2013;Chen and Moore 2014).…”

Leakage and slow allostery limit performance of single drug-sensing aptazyme molecules based on the hammerhead ribozyme

“…3B) than without appended ribozyme (Jucker et al 2003). Yet, coupling of the ligandinduced, localized conformational changes of the aptamer domain through the communication domain into the catalytic core of the hammerhead ribozyme motif (Fig.…”

“…3). The rate constant of the local conformational change in the aptamer domain, as measured in the aptamer mutant, becomes as fast as 63 sec À1 at saturating theophylline concentrations, only about sevenfold slower than in the isolated aptamer (Jucker et al 2003). The observed saturation behavior with Th 1/2 < 0.1 mM suggests that under these conditions the conformational change, not theophylline binding, is rate limiting (Fig.…”

“…3A, upper panel) since nucleotide 279 (Fig. 1A) becomes solvent exposed in the ligand-bound aptamer (Jucker et al 2003). By contrast, the AP fluorescence decreases in the core (A2.1 / AP) mutant upon 10 mM theophylline addition (Fig.…”

Riboswitches: natural SELEXion

“…Global preorganization of the RNA may allow riboswitches to bind with suffi ciently fast kinetics to beat the polymerase. However, studies by Crothers and coworkers on the FMN riboswitch [20] and our group on the purine riboswitch (unpublished data) clearly indicate that natural aptamers bind their ligands on a comparable timescale to that of the theophylline aptamer [21]. Preorganization does not facilitate binding kinetics; instead, riboswitches appear to contain sequence elements that stall the polymerase, giving the RNA suffi cient time for slow events such as ligand binding and secondary-structure rearrangement to occur [20].…”