Direct Measurement of Tertiary Contact Cooperativity in RNA Folding

Abstract

Cooperativity is a central feature of the structure and function of biological macromolecules. It is observed in the folding of proteins to their functional states, in the sharp response of hemoglobin and other proteins to changes in ligand concentration, and in the assembly of macromolecular complexes.Cooperativity in domain formation during protein folding has evolved as a mechanism by which Nature overcomes the difficulty in selectively stabilizing a uniquely folded and functional structure (or small family of structures), among the vast number of partially folded and nonfunctional states that would likely dominate if each of the weak long-range domain contacts were to form independently. This thermodynamic scenario is well-documented for single domain proteins (e.g., refs 1-3 ) and is typically measured using double mutant cycles, analogous to the mutant cycle shown in Figure 1c.Despite its fundamental importance, cooperativity is not always employed in all aspects of protein folding. Regions of proteins often form and break up as units, and these units are sometimes referred to as domains. Further, the cooperativity between domains can vary, from high, for a protein with extensive interfaces and reinforcing interactions, 4 to nonexistent, for a protein like titin with individual domains that are noninteracting "beads on a string". 5 RNA, like proteins, must often fold to distinct three-dimensional structures to carry out biological functions. However, RNA forms stable secondary structure in the absence of tertiary structure, indicating some limits to cooperativity in RNA tertiary folding. Indeed, one might consider folding of an RNA from a preformed secondary structure to a functional tertiary structure as akin to folding of a multidomain protein. The fundamental question then arises: To what extent is there cooperativity in RNA folding?We determined the tertiary contact cooperativity for the independently folding P4-P6 domain derived from the T. thermophila group I intron 6 (Figure 1a,b) using a single molecule fluorescence energy transfer (smFRET) folding assay. The P4-P6 crystal structure in 1996 revealed, for the first time, the side-by-side packing of RNA helices. 7 These helices are connected by a junction (J5/5a) and joined by two regions of tertiary contact, the metal core/ metal core receptor (MC/MCR, Figure 1a, Figure 1a,b, magenta). [6][7][8][9][10] We refer to these regions as "tertiary contacts", and we quantitatively investigate the energetic crosstalk between these tertiary contacts.A thermodynamic scheme for determining the tertiary contact cooperativity in P4-P6 is shown in Figure 1c. The unfolded ensemble (U) comprises the large number of conformations with secondary structure but no tertiary contacts. The unfolded ensemble is in equilibrium with the fully folded state , which has both tertiary contacts formed, and this equilibrium is described by K fold . The two intermediate species, I TL and I MC , have only one tertiary contact formed. In I TL , the tetraloop/receptor contact is formed...

Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

“…1C, MC/MCR, green and TL/TLR, blue). NMR, small angle X-ray scattering, and chemical probing data with WT and mutant P4-P6 RNAs provide evidence for each of these structural states and conditions or mutations that allow each species to predominate (49,51,53,56,(58)(59)(60). Determining the P4-P6 kinetic and thermodynamic framework.…”

“…1C) (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57). Moreover, functional work has uncovered sets of mutations that allow ablation and isolation of these folding intermediates with high confidence (49,51,53,56,(58)(59)(60). The control afforded by this well-behaved, established system provides an opportunity to deepen understanding and develop and test generalizable principles beyond this model system, provided that a detailed framework is in place to provide context for interpretation.…”

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions

“…Herein, we established that physiological concentrations of crowding and cosolutes influence the folding cooperativity of RNA in biological Mg 2+ concentrations. Future studies will be needed to dissect the molecular identity of the cooperativity through such methods as double and triple mutant cycles (Siegfried and Bevilacqua 2009), which we and others have applied under dilute solution conditions (Moody and Bevilacqua 2003;Moody et al 2004;Sattin et al 2008;Behrouzi et al 2012). Moreover, studies will be needed on large, multidomain RNAs to see if these respond differently to crowders and cosolutes.…”

Dissecting electrostatic screening, specific ion binding, and ligand binding in an energetic model for glycine riboswitch folding

“…These efforts represent initial steps in understanding the energetic underpinnings of RNA structure and stability. We emphasize that it will be necessary to go beyond descriptions of RNA folding in terms of the number of states, and even their structures, as a complete understanding of RNA folding will require energetic dissection of each of the features that contribute to and oppose folding, as well as an understanding of the energetic cross-terms-i.e., cooperative and anticooperative terms (Sattin et al 2008). Ultimately, such an understanding will allow prediction of RNA structures and folding energetics and could be used to design new RNAs with unique conformations and properties.…”