Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.

How RNA molecules fold into functional structures is a problem of great significance given the expanding list of essential cellular RNA enzymes and the increasing number of applications of RNA in biotechnology and medicine. A critical step toward solving the RNA folding problem is the characterization of the associated transition states. This is a challenging task in part because the rugged energy landscape of RNA often leads to the coexistence of multiple distinct structural transitions. Here, we exploit single-molecule fluorescence spectroscopy to follow in real time the equilibrium transitions between conformational states of a model RNA enzyme, the hairpin ribozyme. We clearly distinguish structural transitions between effectively noninterchanging sets of unfolded and folded states and characterize key factors defining the transition state of an elementary folding reaction where the hairpin ribozyme's two helical domains dock to make several tertiary contacts. Our singlemolecule experiments in conjunction with site-specific mutations and metal ion titrations show that the two RNA domains are in a contact or close-to-contact configuration in the transition state even though the native tertiary contacts are at most partially formed. Such a compact transition state without well formed tertiary contacts may be a general property of elementary RNA folding reactions. RNA is the key enzymatic component in a number of essential cellular processes, such as translation and splicing (1-4). Aside from these fundamental roles, RNA also finds important applications in modern biotechnology and medicine (5, 6). For example, recent developments in small interfering RNAs, protein-binding RNA aptamers, and target-specific catalytic RNAs suggest that these functional RNAs can serve as effective tools in functional genomics and proteomics and in gene therapy (5,6). This increasing appreciation of RNA as a crucial biopolymer demands more than ever a clear picture of how RNA molecules fold into their native structures, which are vital to their functional properties. A fundamental understanding of RNA folding relies critically on the characterization of the associated folding transition states, i.e., the highest energy states along the reaction coordinates that dictate the transition kinetics. However, the characterization of the transition states of RNA folding lags far behind that of protein folding (7-11), in part because of a more rugged energy landscape for RNA that leads to multiple folding pathways and intermediate states (12)(13)(14)(15)(16)(17)(18)(19), making it difficult to characterize elementary RNA folding transitions. Here, we demonstrate a solution to this problem by using single-molecule fluorescence spectroscopy (20, 21) on a model RNA enzyme, the hairpin ribozyme.Our single-molecule time trajectories unambiguously identify multiple conformational states of the RNA and distinct structural transitions between effectively noninterchanging sets of unfolded and folded states. Using this technique, in conjunction with ...

The hairpin ribozyme is a minimalist paradigm for studying RNA folding and function. In this enzyme, two domains dock by induced fit to form a catalytic core that mediates a specific backbone cleavage reaction. Here, we have fully dissected its reversible reaction pathway, which comprises two structural transitions (docking͞undocking) and a chemistry step (cleavage͞ligation), by applying a combination of single-molecule fluorescence resonance energy transfer (FRET) assays, ensemble cleavage assays, and kinetic simulations. This has allowed us to quantify the effects that modifications of essential functional groups remote from the site of catalysis have on the individual rate constants. We find that all ribozyme variants show similar fractionations into effectively noninterchanging molecule subpopulations of distinct undocking rate constants. This leads to heterogeneous cleavage activity as commonly observed for RNA enzymes. A modification at the domain junction additionally leads to heterogeneous docking. Surprisingly, most modifications not only affect docking͞undocking but also significantly impact the internal chemistry rate constants over a substantial distance from the site of catalysis. We propose that a network of coupled molecular motions connects distant parts of the RNA with its reaction site, which suggests a previously undescribed analogy between RNA and protein enzymes. Our findings also have broad implications for applications such as the action of drugs and ligands distal to the active site or the engineering of allostery into RNA. R NA enzymes (ribozymes) have been recognized as ideal model systems for studying the relationship of structure and function in RNA, because their catalytic activity directly reports the extent of native structure formation (1-3). This provides the basis for powerful modification-interference experiments in which the activity of site-specifically modified ribozymes is compared to the unmodified WT to map functionally important residues of the catalytic core. Such chemical modifications, however, may impact ribozyme function through reaction chemistry, structure formation, or both. Distinguishing these mechanisms has long been an experimental challenge. A typical example is the hairpin ribozyme, derived from the self-cleaving 359-nt negative strand of the tobacco ringspot virus satellite RNA, a member of a family of plant pathogens (4-6). A wealth of modification-interference experiments has helped to define the residues important for function of the minimal two-way junction form of this catalytic RNA, designed as the sequence with highest enzymatic activity in external substrate cleavage (4,5,7,8). Many functional groups of the 24 non-Watson-Crick base-paired nucleotides in the two internal loops of domains A and B were shown to be essential for catalytic activity (Fig. 1A). However, recent crystallographic and biochemical experiments have suggested that only two nucleobases in the ribozyme, G8 and A38, are directly involved in reaction chemistry (Fig. 1 A) (9-12). An...

SummaryRepair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, “open,” and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex.

The yeast SWR1 complex exchanges histone H2A in nucleosomes with Htz1 (H2A.Z in humans). The cryo–electron microscopy structure of the SWR1 complex bound to a nucleosome at 3.6-angstrom resolution reveals details of the intricate interactions between components of the SWR1 complex and its nucleosome substrate. Interactions between the Swr1 motor domains and the DNA wrap at superhelical location 2 distort the DNA, causing a bulge with concomitant translocation of the DNA by one base pair, coupled to conformational changes of the histone core. Furthermore, partial unwrapping of the DNA from the histone core takes place upon binding of nucleosomes to SWR1 complex. The unwrapping, as monitored by single-molecule data, is stabilized and has its dynamics altered by adenosine triphosphate binding but does not require hydrolysis.

The hepatitis delta virus (HDV), an infectious human pathogen and satellite of hepatitis B virus, leads to intensified disease symptoms, including progression to liver cirrhosis. Both the circular RNA genome of HDV and its complementary antigenome contain the same cis-cleaving catalytic RNA motif that plays a crucial role in virus replication. Previously, the high-resolution crystal structure of the product form of a cis-acting genomic HDV ribozyme has been determined, while a trans-acting version of the ribozyme was used to dissect the cleavage reaction pathway. Using fluorescence resonance energy transfer (FRET) on a synthetic trans-cleaving form of the ribozyme, we are able to directly observe substrate binding (at a rate constant k(on) of 7.8 x 10(6) M(-1) min(-1) at pH 7.5, 11 mM MgCl(2), and 25 degrees C) and dissociation (at 0.34 min(-1)). Steady-state and time-resolved FRET experiments in solution and in nondenaturing gels reveal that the substrate (precursor) complex is slightly more compact (by approximately 3 A) than the free ribozyme, yet becomes significantly extended (by approximately 15 A) upon cleavage and product complex formation. We also find that trans cleavage is characterized by a high transition-state entropy (-26 eu). We propose that the significant global conformational change that we observe between the precursor and product structures occurs on the reaction trajectory into a constrained product complex-like transition state. Our observations may present the structural basis of the recently described utilization of intrinsic substrate binding energy to the overall catalytic rate enhancement by the trans-acting HDV ribozyme.

Group II intron ribozymes fold into their native structure by a unique stepwise process that involves an initial slow compaction followed by fast formation of the native state in a Mg 2+ -dependent manner. Single-molecule fluorescence reveals three distinct on-pathway conformations in dynamic equilibrium connected by relatively small activation barriers. From a most stable near-native state, the unobserved catalytically active conformer is reached. This most compact conformer occurs only transiently above 20 mM Mg 2+ and is stabilized by substrate binding, which together explain the slow cleavage of the ribozyme. Structural dynamics increase with increasing Mg 2+ concentrations, enabling the enzyme to reach its active state.

The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.