Structure–Function Analysis from the Outside In: Long-Range Tertiary Contacts in RNA Exhibit Distinct Catalytic Roles

Abstract

The conserved catalytic core of the Tetrahymena group I ribozyme is encircled by peripheral elements. We have carried out a detailed structure-function study of the five long-range tertiary contacts that fasten these distal elements together. Mutational ablation of each of the tertiary contacts destabilizes the folded ribozyme, indicating a role of the peripheral elements in overall stability. Once folded, three of the five tertiary contact mutants exhibit defects in overall catalysis that range from 20- to 100-fold. These and the subsequent results indicate that the structural ring of peripheral elements does not act as a unitary element; rather, individual connections have distinct roles as further revealed by kinetic and thermodynamic dissection of the individual reaction steps. Ablation of P14 or the metal ion core/metal ion core receptor (MC/MCR) destabilizes docking of the substrate-containing P1 helix into tertiary interactions with the ribozyme’s conserved core. In contrast, ablation of the L9/P5 contact weakens binding of the guanosine nucleophile by slowing its association, without affecting P1 docking. The P13 and tetraloop/tetraloop receptor (TL/TLR) mutations had little functional effect and small, local structural changes, as revealed by hydroxyl radical footprinting, whereas the P14, MC/MCR, and L9/P5 mutants show structural changes distal from the mutation site. These changes extended into regions of the catalytic core involved in docking or guanosine binding. Thus, distinct allosteric pathways couple the long-range tertiary contacts to functional sites within the conserved core. This modular functional specialization may represent a fundamental strategy in RNA structure-function interrelationships.

DEAD-box protein CYT-19 is activated by exposed helices in a group I intron RNA

“…Thus, the fraction of substrate that is cleaved in a single turnover reflects the fraction of native ribozyme (40). All of the mutants displayed greater Mg 2+ requirements than the wild-type ribozyme, consistent with previous determinations by hydroxyl radical footprinting and oligonucleotide hybridization (37,39,42,43) (Fig. 2A and Fig.…”

“…To systematically address the roles of RNA stability in chaperone-mediated unfolding, we used a set of previously characterized Tetrahymena ribozyme variants in which the five long-range peripheral tertiary contacts are individually mutated ( Fig. 1) (33,36,37). The mutations include substitutions of the A-rich bulge (ARB) of the metal ion core/metal ion core receptor interaction; the tetraloops L9 and L5b, which form two tetraloop/tetraloop receptor interactions; the L2 loop of the A B "kissing loop" interaction P14; and the L2.1 and L9.1 loops of the P13 kissing loop (Fig.…”

“…The ARB mutant was one of the ribozymes used in the previous study of CYT-19-mediated ribozyme unfolding (28). Footprinting approaches have shown increased Mg 2+ requirements for tertiary folding by each of these and related tertiary contact mutants, indicating decreased global stability (37)(38)(39). Here we focused on the native, catalytically active state of each ribozyme, allowing us to accurately quantify the stability and chaperone-mediated unfolding by measuring oligonucleotide cleavage activity (28,40).…”

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

“…) is several orders of magnitude slower than diffusion (Karbstein and Herschlag 2003), consistent with a model in which the G binding site exists in a configurations that cannot accommodate G and an alternative "open" configuration that can bind G (Hougland et al 2006;Benz-Moy and Herschlag 2011). Multiple structures of group I introns with bound G are available (Adams et al 2004;Guo et al 2004;Golden et al 2005;Stahley and Strobel 2005;Lipchock and Strobel 2008), and all show G forming a base triple interaction with a C-G pair within a "sandwich" of base triples (Supplemental Fig.…”

A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction

“…Just as early quantitative studies of the Tetrahymena ribozyme helped uncover its catalytic properties (e.g., Narlikar et al 1995Narlikar et al , 1997Karbstein and Herschlag 2003;Hougland et al 2006;Benz-Moy and Herschlag 2011) and allowed interpretation of other functional studies and development of a folding framework for this ribozyme (e.g., Herschlag 1999, 2001;Russell et al 2000Russell et al , 2002, our framework for the Azoarcus ribozyme should be similarly empowering. For example, interpretation of reaction burst kinetics used in folding studies (Sinan et al 2011;Behrouzi et al 2012) requires knowledge of the internal equilibrium and of whether substrates are saturating across the range of conditions investigated (described in Wan et al 2009;Sinan et al 2011).…”