Rick Russell

Exploring the folding landscape of a structured RNA

Structured RNAs achieve their active states by traversing complex, multidimensional energetic landscapes. Here we probe the folding landscape of the Tetrahymena ribozyme by using a powerful approach: the folding of single ribozyme molecules is followed beginning from distinct regions of the folding landscape. The experiments, combined with small-angle x-ray scattering results, show that the landscape contains discrete folding pathways. These pathways are separated by large free-energy barriers that prevent interconversion between them, indicating that the pathways lie in deep channels in the folding landscape. Chemical protection and mutagenesis experiments are then used to elucidate the structural features that determine which folding pathway is followed. Strikingly, a specific long-range tertiary contact can either help folding or hinder folding, depending on when it is formed during the process. Together these results provide an unprecedented view of the topology of an RNA folding landscape and the RNA structural features that underlie this multidimensional landscape.

Rapid compaction during RNA folding

We have used small angle x-ray scattering and computer simulations with a coarse-grained model to provide a time-resolved picture of the global folding process of the Tetrahymena group I RNA over a time window of more than five orders of magnitude. A substantial phase of compaction is observed on the low millisecond timescale, and the overall compaction and global shape changes are largely complete within one second, earlier than any known tertiary contacts are formed. This finding indicates that the RNA forms a nonspecifically collapsed intermediate and then searches for its tertiary contacts within a highly restricted subset of conformational space. The collapsed intermediate early in folding of this RNA is grossly akin to molten globule intermediates in protein folding.

DEAD-box proteins can completely separate an RNA duplex using a single ATP

DEAD-box proteins are ubiquitous in RNA metabolism and use ATP to mediate RNA conformational changes. These proteins have been suggested to use a fundamentally different mechanism from the related DNA and RNA helicases, generating local strand separation while remaining tethered through additional interactions with structured RNAs and RNA-protein (RNP) complexes. Here, we provide a critical test of this model by measuring the number of ATP molecules hydrolyzed by DEAD-box proteins as they separate short RNA helices characteristic of structured RNAs (6–11 bp). We show that the DEAD-box protein CYT-19 can achieve complete strand separation using a single ATP, and that 2 related proteins, Mss116p and Ded1p, display similar behavior. Under some conditions, considerably <1 ATP is hydrolyzed per separation event, even though strand separation is strongly dependent on ATP and is not supported by the nucleotide analog AMP-PNP. Thus, ATP strongly enhances strand separation activity even without being hydrolyzed, most likely by eliciting or stabilizing a protein conformation that promotes strand separation, and AMP-PNP does not mimic ATP in this regard. Together, our results show that DEAD-box proteins can disrupt short duplexes by using a single cycle of ATP-dependent conformational changes, strongly supporting and extending models in which DEAD-box proteins perform local rearrangements while remaining tethered to their target RNAs or RNP complexes. This mechanism may underlie the functions of DEAD-box proteins by allowing them to generate local rearrangements without disrupting the global structures of their targets.

RNA helicase proteins as chaperones and remodelers

Superfamily 2 helicase proteins are ubiquitous in RNA biology and have an extraordinarily broad set of functional roles. Central among these roles are the promotion of rearrangements of structured RNAs and the remodeling of ribonucleoprotein complexes (RNPs), allowing formation of native RNA structure or progression through a functional cycle of structures. Although all superfamily 2 helicases share a conserved helicase core, they are divided evolutionarily into several families, and it is principally proteins from three families, the DEAD-box, DEAH/RHA, and Ski2-like families, that function to manipulate structured RNAs and RNPs. Strikingly, there are emerging differences in the mechanisms of these proteins, both between families and within the largest family (DEAD-box), and these differences appear to be tuned to their RNA or RNP substrates and their specific roles. This review outlines basic mechanistic features of the three families and surveys individual proteins and the current understanding of their biological substrates and mechanisms.

RNA misfolding and the action of chaperones.

RNA folds to a myriad of three-dimensional structures and performs an equally diverse set of functions. The ability of RNA to fold and function in vivo is all the more remarkable because, in vitro, RNA has been shown to have a strong propensity to adopt misfolded, non-functional conformations. A principal factor underlying the dominance of RNA misfolding is that local RNA structure can be quite stable even in the absence of enforcing global tertiary structure. This property allows non-native structure to persist, and it also allows native structure to form and stabilize non-native contacts or non-native topology. In recent years it has become clear that one of the central reasons for the apparent disconnect between the capabilities of RNA in vivo and its in vitro folding properties is the presence of RNA chaperones, which facilitate conformational transitions of RNA and therefore mitigate the deleterious effects of RNA misfolding. Over the past two decades, it has been demonstrated that several classes of non-specific RNA binding proteins possess profound RNA chaperone activity in vitro and when overexpressed in vivo, and at least some of these proteins appear to function as chaperones in vivo. More recently, it has been shown that certain DExD/H-box proteins function as general chaperones to facilitate folding of group I and group II introns. These proteins are RNA-dependent ATPases and have RNA helicase activity, and are proposed to function by using energy from ATP binding and hydrolysis to disrupt RNA structure and/or to displace proteins from RNA-protein complexes. This review outlines experimental studies that have led to our current understanding of the range of misfolded RNA structures, the physical origins of RNA misfolding, and the functions and mechanisms of putative RNA chaperone proteins.

DEAD-box proteins as RNA helicases and chaperones.

DEAD‐box proteins are ubiquitous in RNA‐mediated processes and function by coupling cycles of ATP binding and hydrolysis to changes in affinity for single‐stranded RNA. Many DEAD‐box proteins use this basic mechanism as the foundation for a version of RNA helicase activity, efficiently separating the strands of short RNA duplexes in a process that involves little or no translocation. This activity, coupled with mechanisms to direct different DEAD‐box proteins to their physiological substrates, allows them to promote RNA folding steps and rearrangements and to accelerate remodeling of RNA–protein complexes. This review will describe the properties of DEAD‐box proteins as RNA helicases and the current understanding of how the energy from ATPase activity is used to drive the separation of RNA duplex strands. It will then describe how the basic biochemical properties allow some DEAD‐box proteins to function as chaperones by promoting RNA folding reactions, with a focus on the self‐splicing group I and group II intron RNAs. WIREs RNA 2011 2 135–152 DOI: 10.1002/wrna.50

Kinetic redistribution of native and misfolded RNAs by a DEAD-box chaperone

DExD/H-box proteins are ubiquitously involved in RNA-mediated processes and use ATP to accelerate conformational changes in RNA. However, their mechanisms of action, and what determines which RNA species are targeted, are not well understood. Here we show that the DExD/H-box protein CYT-19, a general RNA chaperone, mediates ATP-dependent unfolding of both the native conformation and a long-lived misfolded conformation of a group I catalytic RNA with efficiencies that depend on the stabilities of the RNA species but not on specific structural features. CYT-19 then allows the RNA to refold, changing the distribution from equilibrium to kinetic control. Because misfolding is favoured kinetically, conditions that allow unfolding of the native RNA yield large increases in the population of misfolded species. Our results suggest that DExD/H-box proteins act with sufficient breadth and efficiency to allow structured RNAs to populate a wider range of conformations than would be present at equilibrium. Thus, RNAs may face selective pressure to stabilize their active conformations relative to inactive ones to avoid significant redistribution by DExD/H-box proteins. Conversely, RNAs whose functions depend on forming multiple conformations may rely on DExD/H-box proteins to increase the populations of less stable conformations, thereby increasing their overall efficiencies.

Nonspecific binding to structured RNA and preferential unwinding of an exposed helix by the CYT-19 protein, a DEAD-box RNA chaperone

We explore the interactions of CYT-19, a DExD/H-box protein that functions in folding of group I RNAs, with a well characterized misfolded species of the Tetrahymena ribozyme. Consistent with its function, CYT-19 accelerates refolding of the misfolded RNA to its native state. Unexpectedly, CYT-19 performs another reaction much more efficiently; it unwinds the 6-bp P1 duplex formed between the ribozyme and its oligonucleotide substrate. Furthermore, CYT-19 performs this reaction 50-fold more efficiently than it unwinds the same duplex free in solution, suggesting that it forms additional interactions with the ribozyme, most likely using a distinct RNA binding site from the one responsible for unwinding. This site can apparently bind double-stranded RNA, as attachment of a simple duplex adjacent to P1 recapitulates much of the activation provided by the ribozyme. Unwinding the native P1 duplex does not accelerate refolding of the misfolded ribozyme, implying that CYT-19 can disrupt multiple contacts on the RNA, consistent with its function in folding of multiple RNAs. Further experiments showed that the P1 duplex unwinding activity is virtually the same whether the ribozyme is misfolded or native but is abrogated by formation of tertiary contacts between the P1 duplex and the body of the ribozyme. Together these results suggest a mechanism for CYT-19 and other general DExD/H-box RNA chaperones in which the proteins bind to structured RNAs and efficiently unwind loosely associated duplexes, which biases the proteins to disrupt nonnative base pairs and gives the liberated strands an opportunity to refold.

Solution structures of DEAD-box RNA chaperones reveal conformational changes and nucleic acid tethering by a basic tail

The mitochondrial DEAD-box proteins Mss116p of Saccharomyces cerevisiae and CYT-19 of Neurospora crassa are ATP-dependent helicases that function as general RNA chaperones. The helicase core of each protein precedes a C-terminal extension and a basic tail, whose structural role is unclear. Here we used small-angle X-ray scattering to obtain solution structures of the full-length proteins and a series of deletion mutants. We find that the two core domains have a preferred relative orientation in the open state without substrates, and we visualize the transition to a compact closed state upon binding RNA and adenosine nucleotide. An analysis of complexes with large chimeric oligonucleotides shows that the basic tails of both proteins are attached flexibly, enabling them to bind rigid duplex DNA segments extending from the core in different directions. Our results indicate that the basic tails of DEAD-box proteins contribute to RNA-chaperone activity by binding nonspecifically to large RNA substrates and flexibly tethering the core for the unwinding of neighboring duplexes.

Toward a molecular understanding of RNA remodeling by DEAD-box proteins

DEAD-box proteins are superfamily 2 helicases that function in all aspects of RNA metabolism. They employ ATP binding and hydrolysis to generate tight, yet regulated RNA binding, which is used to unwind short RNA helices non-processively and promote structural transitions of RNA and RNA-protein substrates. In the last few years, substantial progress has been made toward a detailed, quantitative understanding of the structural and biochemical properties of DEAD-box proteins. Concurrently, progress has been made toward a physical understanding of the RNA rearrangements and folding steps that are accelerated by DEAD-box proteins in model systems. Here, we review the recent progress on both of these fronts, focusing on the mitochondrial DEAD-box proteins Mss116 and CYT-19 and their mechanisms in promoting the splicing of group I and group II introns.