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

Abstract

Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg 2+ ion concentrations are low, K + concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo-like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg 2+ (0.5-2 mM) and K + (140 mM) if the solution is supplemented with physiological amounts (∼20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperaturedependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution.

Bridging the gap betweenin vitroandin vivoRNA folding

“…Several recent studies focused on mimicking aspects of the in vivo environment in vitro ; conditions referred to herein as ‘ in vivo-like ’ conditions (Figure 3). Effects of such conditions as cellular concentrations of monovalent and divalent ions and molecular crowding agents on the folding of RNAs have been a theme in a number of recent studies (Desai et al, 2014; Dupuis et al, 2014; Nakano et al, 2015; Paudel & Rueda, 2014; Strulson et al, 2014; Tyrrell et al, 2015). Experiments under these in vivo-like conditions have the potential to bridge our understanding of observations made in vitro and in vivo .…”

“…Various methods, including UV melts, SAXS, kinetic techniques, and smFRET, have been used to study RNA under these in vivo-like conditions. Several studies have shown that synthetic crowding agents affect the thermodynamics and function of several RNAs (Dupuis et al, 2014; Kilburn et al, 2013; Kilburn et al, 2010; Lambert et al, 2010; Strulson et al, 2014). Findings of these studies are that RNAs fold cooperatively, structure becomes compact, and ribozymes cleave faster under in vivo-like conditions (Kilburn et al, 2013; Nakano et al, 2009; Strulson et al, 2014; Strulson et al, 2013).…”

Structures and functions in the crowded nucleus: new biophysical insights

“…-the effects of crowding on the structure in vivo of chromatin which contains DNA in conformations other than the classical B-form double helix, such as the DNA in telomeres whose conformation in vitro is strongly influenced by crowding [80]; -the consequences of crowding for the structures of RNAs and ribonucleoproteins in vivo; the folding and stability of RNA in vitro are enhanced significantly by crowding [81][82][83]; -loops in chromatin fibers in vivo are usually thought to be stabilized by proteins such as cohesin (for example [84]). Reports that nucleosomes which contain identical DNA sequences can self-associate preferentially [85] raise the possibility that similar interactions could contribute to the formation and stabilization of loops [86].…”

A Highly Coupled Network of Tertiary Interactions in the SAM-I Riboswitch and Their Role in Regulatory Tuning

“…Coupled tertiary interactions that form early in the folding process have been observed in the group I self-splicing intron, which promote proper RNA folding by suppressing nonnative structures [11]. The network of peripheral tertiary elements might be more important within the cellular environment as RNA tertiary interactions are strengthened relative to secondary interactions within macromolecular crowders and cosolutes [12]. The highly coupled nature of tertiary structure in RNA also likely makes it poorly suited for modulation of activity such as regulatory tuning.…”

“…The coupled and cooperative tertiary interactions assist the RNA in traversing a rugged folding landscape. Further, studies have recently started to focus on RNA structure and folding under conditions mimicking physiological conditions [12–14] or co-transcriptional constraints [15–17]. Transcriptional riboswitches are an ideal model system for studying RNA folding under co-transcriptional constraints because a regulatory decision via transcriptional termination or read-through must be made at the time the RNA polymerase reaches the poly-uridine tract of the intrinsic (rho-independent) terminator [18].…”