Alternative spliceosome assembly pathways revealed by single-molecule fluorescence microscopy.

Removal of introns from nascent transcripts (pre-mRNAs) by the spliceosome is an essential step in eukaryotic gene expression. Previous studies have suggested that the earliest steps in spliceosome assembly in yeast are highly ordered and the stable recruitment of U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site necessarily precedes recruitment of U2 snRNP to the branch site to form the "prespliceosome." Here, using colocalization single-molecule spectroscopy to follow initial spliceosome assembly on eight different S. cerevisiae pre-mRNAs, we demonstrate that active yeast spliceosomes can form by both U1-first and U2-first pathways. Both assembly pathways yield prespliceosomes functionally equivalent for subsequent U5·U4/U6 tri-snRNP recruitment and for intron excision. Although fractional flux through the two pathways varies on different introns, both are operational on all introns studied. Thus, multiple pathways exist for assembling functional spliceosomes. These observations provide insight into the mechanisms of cross-intron coordination of initial spliceosome assembly.

Hydroxyl-radical footprinting to probe equilibrium changes in RNA tertiary structure.

Hydroxyl-radical footprinting utilizes the ability of a highly reactive species to nonspecifically cleave the solvent accessible regions of a nucleic acid backbone. Thus, changes in a nucleic acids structure can be probed either as a function of time or of a reagent's concentration. When combined with techniques that allow single nucleotide resolution of the resulting fragments, footprinting experiments provide richly detailed information about local changes in tertiary structure of a nucleic acid accompanying its folding or ligand binding. In this chapter, we present two protocols of equilibrium hydroxyl-radical footprinting based on peroxidative and oxidative Fenton chemistry and discuss how to adjust the Fenton reagent concentrations for a specific experimental condition. We also discuss the choice of the techniques to separate the reaction products and specifics of the data analysis for equilibrium footprinting experiments. Protocols addressing the use of peroxidative Fenton chemistry for time-resolved studies have been published [Schlatterer and Brenowitz, 2009. Methods; Shcherbakova and Brenowitz, 2008. Nat. Protoc.3(2), 288-302; Shcherbakova et al., 2006. Nucleic Acids Res.34(6), e48; Shcherbakova et al., 2007. Methods Cell Biol.84, 589-615].

Rapid quantification and analysis of kinetic •OH radical footprinting data using SAFA.

The use of highly reactive chemical species to probe the structure and dynamics of nucleic acids is greatly simplified by software that enables rapid quantification of the gel images that result from these experiments. Semiautomated footprinting analysis (SAFA) allows a user to quickly and reproducibly quantify a chemical footprinting gel image through a series of steps that rectify, assign, and integrate the relative band intensities. The output of this procedure is raw band intensities that report on the relative reactivity of each nucleotide with the chemical probe. We describe here how to obtain these raw band intensities using SAFA and the subsequent normalization and analysis procedures required to process these data. In particular, we focus on analyzing time-resolved hydroxyl radical ((•)OH) data, which we use to monitor the kinetics of folding of a large RNA (the L-21 T. thermophila group I intron). Exposing the RNA to bursts of (•)OH radicals at specific time points during the folding process monitors the time progress of the reaction. Specifically, we identify protected (nucleotides that become inaccessible to the (•)OH radical probe when folded) and invariant (nucleotides with constant accessibility to the (•)OH probe) residues that we use for monitoring and normalization of the data. With this analysis, we obtain time-progress curves from which we determine kinetic rates of folding. We also report on a data visualization tool implemented in SAFA that allows users to map data onto a secondary structure diagram.

Energy barriers, pathways, and dynamics during folding of large, multidomain RNAs.

Large, multidomain RNA molecules are generally thought to fold following multiple pathways down rugged landscapes populated with intermediates and traps. A challenge to understanding RNA folding reactions is the complex relationships that exist between the structure of the RNA and its folding landscape. The identification of intermediate species that populate folding landscapes and characterization of elements of their structures are the key components to solving the RNA folding problem. This review explores recent studies that characterize the dominant pathways by which RNA folds, structural and dynamic features of intermediates that populate the folding landscape, and the energy barriers that separate the distinct steps of the folding process.

Function and structural organization of Mot1 bound to a natural target promoter.

Mot1 is an essential, conserved TATA-binding protein (TBP)-associated factor in Saccharomyces cerevisiae and a member of the Snf2/Swi2 ATPase family. Mot1 uses ATP hydrolysis to displace TBP from DNA, an activity that can be readily reconciled with its global role in gene repression. Less well understood is how Mot1 directly activates gene expression. It has been suggested that Mot1-mediated activation can occur by displacement of inactive TBP-containing complexes from promoters, thereby permitting assembly of functional transcription complexes. Mot1 may also activate transcription by other mechanisms that have not yet been defined. A gap in our understanding has been the absence of biochemical information related to the activity of Mot1 on natural target genes. Using URA1 as a model Mot1-activated promoter, we show striking differences in the way that both TBP and Mot1 interact with DNA compared with other model DNA substrates analyzed previously. These differences are due at least in part to the propensity of TBP alone to bind to the URA1 promoter in the wrong orientation to direct appropriate assembly of the URA1 preinitiation complex. The results suggest that Mot1-mediated activation of URA1 transcription involves at least two steps, one of which is the removal of TBP bound to the promoter in the opposite orientation required for URA1 transcription.

High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis.

The use of capillary electrophoresis with fluorescently labeled nucleic acids revolutionized DNA sequencing, effectively fueling the genomic revolution. We present an application of this technology for the high-throughput structural analysis of nucleic acids by chemical and enzymatic mapping ('footprinting'). We achieve the throughput and data quality necessary for genomic-scale structural analysis by combining fluorophore labeling of nucleic acids with novel quantitation algorithms. We implemented these algorithms in the CAFA (capillary automated footprinting analysis) open-source software that is downloadable gratis from https://simtk.org/home/cafa. The accuracy, throughput and reproducibility of CAFA analysis are demonstrated using hydroxyl radical footprinting of RNA. The versatility of CAFA is illustrated by dimethyl sulfate mapping of RNA secondary structure and DNase I mapping of a protein binding to a specific sequence of DNA. Our experimental and computational approach facilitates the acquisition of high-throughput chemical probing data for solution structural analysis of nucleic acids.

Monitoring structural changes in nucleic acids with single residue spatial and millisecond time resolution by quantitative hydroxyl radical footprinting.

Hydroxyl radical (.OH) footprinting provides comprehensive site-specific quantitative information about the structural changes associated with macromolecular folding, interactions and ligand binding. 'Fast Fenton' footprinting is a laboratory-based method for time-resolved .OH footprinting capable of millisecond time resolution readily applicable to DNA and RNA. This protocol utilizes inexpensive chemical reagents (H2O2, Fe(NH4)2(SO4)2, EDTA, thiourea or ethanol) and widely available quench-flow mixers to reveal transient, often short-lived, intermediate states of complex biochemical processes. We describe a protocol developed to study RNA folding that can be readily tailored to particular applications. Once familiar with quench-flow mixer operation and its calibration, nucleic acid labeling and the conduct of a dose-response experiment, a single kinetic experiment of 30 time points takes about 1 h to perform. Sample processing and separation of the .OH reaction products takes several hours. Data analysis can take 45 min to several weeks depending on the depth of analysis conducted.

Following molecular transitions with single residue spatial and millisecond time resolution.

"Footprinting" describes assays in which ligand binding or structure formation protects polymers such as nucleic acids and proteins from either cleavage or modification; footprinting allows the accessibility of individual residues to be mapped in solution. Equilibrium and time-dependent footprinting links site-specific structural information with thermodynamic and kinetic transitions, respectively. The hydroxyl radical (*OH) is a uniquely insightful footprinting probe by virtue of it being among the most reactive chemical oxidants; it reports the solvent accessibility of reactive sites on macromolecules with as fine as a single residue resolution. A novel method of millisecond time-resolved *OH footprinting is presented based on the Fenton reaction, Fe(II) + H(2)O(2) --> Fe(III) + *OH + OH(-). It is implemented using a standard three-syringe quench-flow mixer. The utility of this method is demonstrated by its application to the studies on RNA folding. Its applicability to a broad range of biological questions involving the function of DNA, RNA, and proteins is discussed.

Distinct contribution of electrostatics, initial conformational ensemble, and macromolecular stability in RNA folding.

We distinguish the contribution of the electrostatic environment, initial conformational ensemble, and macromolecular stability on the folding mechanism of a large RNA using a combination of time-resolved "Fast Fenton" hydroxyl radical footprinting and exhaustive kinetic modeling. This integrated approach allows us to define the folding landscape of the L-21 Tetrahymena thermophila group I intron structurally and kinetically from its earliest steps with unprecedented accuracy. Distinct parallel pathways leading the RNA to its native form upon its Mg(2+)-induced folding are observed. The structures of the intermediates populating the pathways are not affected by variation of the concentration and type of background monovalent ions (electrostatic environment) but are altered by a mutation that destabilizes one domain of the ribozyme. Experiments starting from different conformational ensembles but folding under identical conditions show that whereas the electrostatic environment modulates molecular flux through different pathways, the initial conformational ensemble determines the partitioning of the flux. This study showcases a robust approach for the development of kinetic models from collections of local structural probes.

Fast Fenton footprinting: a laboratory-based method for the time-resolved analysis of DNA, RNA and proteins.

'Footprinting' describes assays in which ligand binding or structure formation protects polymers such as nucleic acids and proteins from either cleavage or modification; footprinting allows the accessibility of individual residues to be mapped in solution. Equilibrium and time-dependent footprinting links site-specific structural information with thermodynamic and kinetic transitions. The hydroxyl radical (*OH) is a particularly valuable footprinting probe by virtue of it being among the most reactive of chemical oxidants; it reports the solvent accessibility of reactive sites on macromolecules with as fine as a single residue resolution. A novel method of millisecond time-resolved .OH footprinting has been developed based on the Fenton reaction, Fe(II) + H2O2 --> Fe(III) + *OH + OH-. This method can be implemented in laboratories using widely available three-syringe quench flow mixers and inexpensive reagents to study local changes in the solvent accessibility of DNA, RNA and proteins associated with their biological function.

Local kinetic measures of macromolecular structure reveal partitioning among multiple parallel pathways from the earliest steps in the folding of a large RNA molecule.

At the heart of the RNA folding problem is the number, structures, and relationships among the intermediates that populate the folding pathways of most large RNA molecules. Unique insight into the structural dynamics of these intermediates can be gleaned from the time-dependent changes in local probes of macromolecular conformation (e.g. reports on individual nucleotide solvent accessibility offered by hydroxyl radical (()OH) footprinting). Local measures distributed around a macromolecule individually illuminate the ensemble of separate changes that constitute a folding reaction. Folding pathway reconstruction from a multitude of these individual measures is daunting due to the combinatorial explosion of possible kinetic models as the number of independent local measures increases. Fortunately, clustering of time progress curves sufficiently reduces the dimensionality of the data so as to make reconstruction computationally tractable. The most likely folding topology and intermediates can then be identified by exhaustively enumerating all possible kinetic models on a super-computer grid. The folding pathways and measures of the relative flux through them were determined for Mg(2+) and Na(+)-mediated folding of the Tetrahymena thermophila group I intron using this combined experimental and computational approach. The flux during Mg(2+)-mediated folding is divided among numerous parallel pathways. In contrast, the flux during the Na(+)-mediated reaction is predominantly restricted through three pathways, one of which is without detectable passage through intermediates. Under both conditions, the folding reaction is highly parallel with no single pathway accounting for more than 50% of the molecular flux. This suggests that RNA folding is non-sequential under a variety of different experimental conditions even at the earliest stages of folding. This study provides a template for the systematic analysis of the time-evolution of RNA structure from ensembles of local measures that will illuminate the chemical and physical characteristics of each step in the process. The applicability of this analysis approach to other macromolecules is discussed.

Concordant exploration of the kinetics of RNA folding from global and local perspectives.

Time-resolved small-angle X-ray scattering (SAXS) with millisecond time-resolution reveals two discrete phases of global compaction upon Mg2+-mediated folding of the Tetrahymena thermophila ribozyme. Electrostatic relaxation of the RNA occurs rapidly and dominates the first phase of compaction during which the observed radius of gyration (R(g)) decreases from 75 angstroms to 55 angstroms. A further decrease in R(g) to 45 angstroms occurs in a well-defined second phase. An analysis of mutant ribozymes shows that the latter phase depends upon the formation of long-range tertiary contacts within the P4-P6 domain of the ribozyme; disruption of the three remaining long-range contacts linking the peripheral helices has no effect on the 55-45 angstroms compaction transition. A better understanding of the role of specific tertiary contacts in compaction was obtained by concordant time-resolved hydroxyl radical (OH) analyses that report local changes in the solvent accessibility of the RNA backbone. Comparison of the global and local measures of folding shows that formation of a subset of native tertiary contacts (i.e. those defining the ribozyme core) can occur within a highly compact ensemble whose R(g) is close to that of the fully folded ribozyme. Analyses of additional ribozyme mutants and reaction conditions establish the generality of the rapid formation of a partially collapsed state with little to no detectable tertiary structure. These studies directly link global RNA compaction with formation of tertiary structure as the molecule acquires its biologically active structure, and underscore the strong dependence on salt of both local and global measures of folding kinetics.

Perturbation of the hierarchical folding of a large RNA by the destabilization of its Scaffold's tertiary structure.

The P4-P6 domain serves as a scaffold against which the periphery and catalytic core organize and fold during Mg2+-mediated folding of the Tetrahymena thermophila ribozyme. The most prominent structural motif of the P4-P6 domain is the tetraloop-tetraloop receptor interaction which "clamps" the distal parts of its hairpin-like structure. Destabilization of the tertiary structure of the P4-P6 domain by perturbation of the tetraloop-tetraloop receptor interaction alters the Mg2+-mediated folding pathway. The folding hierarchy of P5c approximately P4-P6 > periphery > catalytic core that is a striking attribute of the folding of the wild-type RNA is abolished. The initial steps in folding of the mutant RNA are > or =50-fold faster than those of the wild-type ribozyme with the earliest observed tertiary contacts forming around regions known to specifically bind Mg2+. The interaction between the mutant tetraloop and the tetraloop receptor appears coincidently with slowly forming catalytic core tertiary contacts. Thus, the stability conferred upon the P4-P6 domain by the tetraloop-tetraloop receptor interaction dictates the preferred folding pathway by stabilizing an early intermediate. A sub-denaturing concentration of urea diminishes the early barrier to folding the wild-type ribozyme along with complex effects on the subsequent steps of folding the wild-type and mutant RNA.

Monovalent ion-mediated folding of the Tetrahymena thermophila ribozyme.

The time-course of monovalent cation-induced folding of the L-21 Sca1 Tetrahymena thermophila ribozyme and a selected mutant was quantitatively followed using synchrotron X-ray (.OH) footprinting. Initiating folding by increasing the concentration of either Na+ or K+ to 1.5M from an initial condition of approximately 0.008 M Na+ at 42 degrees C resulted in the complete formation of tertiary contacts within the P5abc subdomain and between the peripheral helices within the dead time of our measurements (k>50 s(-1)). These results contrast with folding rates of 2-0.2 s(-1) previously observed for formation of these contacts in 10mM Mg2+ from the same initial condition. Thus, the initial formation of native tertiary contacts is inhibited by divalent but not monovalent cations. The native contacts within the catalytic core form without a detectable burst phase at rates of 0.4-1.0 s(-1) in a manner reminiscent of the Mg2+-dependent folding behavior, although tenfold faster. The tertiary interactions stabilizing the catalytic core interaction with P4-P6 and P2.1, as well as one of the protections internal for the P4-P6 domain, display progress curves with appreciable burst amplitudes and a phase comparable in rate to that of the catalytic core. That the slow folding of the ribozyme's core is a consequence of the alt-P3 secondary structure is shown by the 100% burst phase amplitudes that are observed for folding of the U273A mutant ribozyme within which the native secondary structure (P3) is strengthened. Thus, formation of a misfolded intermediate(s) resulting from the alt-P3 secondary structure is independent of ion valency while the rate at which the respective intermediates are resolved is sensitive to ion valency. The overall portrait painted by these results is that ion valency differentially affects steps in the folding process and that folding in monovalent ion alone for the U273A mutant Tetrahymena ribozyme is fast and direct.