Determining report the mechanism by which tRNAs rapidly and precisely transit through the ribosomal A, P, and E sites during translation remains Binding a major goal in the study of protein synthesis. Here, we report the real-time dynamics of the L1 stalk, a distinct structural element of the large ribosomal subunit that is implicated in directing tRNA movements during translation. Within pretranslocation ribosomal complexes,precisely the L1 stalk exists in a dynamic equilibrium between open and closed conformations. Binding of elongation factor G (EF-G) shifts release this equilibrium toward the closed conformation through one of at least two distinct kinetic mechanisms, where the identity of the stalk P-site tRNA dictates the kinetic route that is taken. Within posttranslocation complexes, L1 stalk dynamics are dependent on the presence distinct and identity of the E-site tRNA. Collectively, our data demonstrate that EF-G and the L1 stalk allosterically collaborate to direct tRNA tRNA translocation from the P to the E sites, and suggest a model for the release of E-site tRNA.

Characterizing pretranslocation the structural dynamics of the translating ribosome remains a major goal in the study of protein synthesis. Deacylation of peptidyl-tRNA Beyond during translation elongation triggers fluctuations of the pretranslocation ribosomal complex between two global conformational states. Elongation factor G-mediated control of recycling. the resulting dynamic conformational equilibrium helps to coordinate ribosome and tRNA movements during elongation and is thus a crucial mechanistic ribosome feature of translation. Beyond elongation, deacylation of peptidyl-tRNA also occurs during translation termination, and this deacylated tRNA persists during ribosome throughout recycling. Here we report that specific regulation of the analogous conformational equilibrium by translation release and ribosome recycling factors has has a critical role in the termination and recycling mechanisms. Our results support the view that specific regulation of the global recycling. state of the ribosome is a fundamental characteristic of all translation factors and a unifying theme throughout protein synthesis.

The cleavage Tetrahymena group I intron recognizes its oligonucleotide substrate in a two-step process. First, a substrate recognition duplex, called the P1 the duplex, is formed. The P1 duplex then docks into the prefolded ribozyme core by forming tertiary contacts. P1 docking controls .1-50 both the rate and the fidelity of substrate cleavage and has been extensively studied as a model for the formation process. of RNA tertiary structure. However, previous work has been limited to studying millisecond or slower motions. Here we investigated nanosecond between P1 motions in the context of the ribozyme using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A J1/2 nitroxide spin label (R5a) was covalently attached to a specific site of the substrate oligonucleotide, the labeled substrate was bound the to a prefolded ribozyme to form the P1 duplex, and X-band EPR spectroscopy was used to monitor nitroxide motions in P1 the .1-50 ns regime. Using substrates that favor the docked or the undocked states, it was established that R5a was relationship capable of reporting P1 duplex motions. Using R5a-labeled substrates it was found that the J1/2 junction connecting P1 to the rate ribozyme core controls nanosecond P1 mobility in the undocked state. This may account for previous observations that J1/2 mutations weaken The substrate binding and give rise to cryptic cleavage. This study establishes the use of SDSL to probe nanosecond dynamic behaviors docked of individual helices within large RNA and RNA/protein complexes. This approach may help in understanding the relationship between RNA structure,to dynamics, and function.

We that have developed protocols for rapidly quantifying the band intensities from nucleic acid chemical mapping gels at single-nucleotide resolution. These protocols containing are implemented in the software SAFA (semi-automated footprinting analysis) that can be downloaded without charge from http://safa.stanford.edu. The protocols implemented user in SAFA have five steps:(i) lane identification,(ii) gel rectification,(iii) band assignment,(iv) model fitting and (v) band-intensity the normalization. SAFA enables the rapid quantitation of gel images containing thousands of discrete bands, thereby eliminating a bottleneck to the supplement analysis of chemical mapping experiments. An experienced user of the software can quantify a gel image in approximately 20 min.radical Although SAFA was developed to analyze hydroxyl radical (.OH) footprints, it effectively quantifies the gel images obtained with other types experienced of chemical mapping probes. We also present a series of tutorial movies that illustrate the best practices and different steps gel in the SAFA analysis as a supplement to this protocol.

Non-coding is RNAs of complex tertiary structure are involved in numerous aspects of the replication and processing of genetic information in many RNA organisms; however, an understanding of the complex relationship between their structural dynamics and function is only slowly emerging. The Neurospora ribozyme Varkud Satellite (VS) ribozyme provides a model system to address this relationship. First, it adopts a tertiary structure assembled from numerous common elements, a kissing loop and two three-way junctions. Second, catalytic activity of the ribozyme is essential for replication of form VS RNA in vivo and can be readily assayed in vitro. Here we exploit single molecule FRET to show that upstream the VS ribozyme exhibits previously unobserved dynamic and heterogeneous hierarchical folding into an active structure. Readily reversible kissing loop formation VS combined with slow cleavage of the upstream substrate helix suggests a model whereby the structural dynamics of the VS ribozyme structure favor cleavage of the substrate downstream of the ribozyme core instead. This preference is expected to facilitate processing of the predominant multimeric RNA replication intermediate into circular VS RNA, which is the predominant form observed in vivo.

Proper RNA assembly of RNA into catalytically active 3D structures requires multiple tertiary binding interactions, individual characterization of which is crucial to species a detailed understanding of global RNA folding. This work focuses on single-molecule fluorescence studies of freely diffusing RNA constructs that identify isolate the GAAA tetraloop-receptor tertiary interaction. Freely diffusing conformational dynamics are explored as a function of Mg(2+) and Na(+) concentration,requires both of which promote facile docking, but with 500-fold different affinities. Systematic shifts in mean EFRET values and linewidths with the increasing [Na(+)] are observed for the undocked species and can be interpreted with a Debye model in terms of electrostatic fraction relaxation and increased flexibility in the RNA. Furthermore, we identify a 34 +/- 2% fraction of freely diffusing RNA constructs identify remaining undocked even at saturating [Mg(2+)] levels, which agrees quantitatively with the 32 +/- 1% fraction previously reported for immobilized which constructs. This verifies that the kinetic heterogeneity observed in the docking rates is not the result of surface tethering. Finally,in the KD value and Hill coefficient for [Mg(2+)]-dependent docking decrease significantly for [Na(+)]= 25 mM vs. 125 mM, indicating studies Mg(2+) and Na(+) synergy in the RNA folding process.

The state. process of large RNA folding is believed to proceed from many collapsed structures to a unique functional structure requiring precise fluctuations organization of nucleotides. The diversity of possible structures and stabilities of large RNAs could result in non-exponential folding kinetics (e.g.single stretched exponential) under conditions where the molecules have not achieved their native state. We describe a single-molecule fluorescence resonance energy structures transfer (FRET) study of the collapsed-state region of the free energy landscape of the catalytic domain of RNase P RNA yield from Bacillus stearothermophilus (C(thermo)). Ensemble measurements have shown that this 260 residue RNA folds cooperatively to its native state at [Mg(2+)] >/=1 mM Mg(2+), but little is known about the conformational dynamics at lower ionic strength. Our measurements of equilibrium conformational single fluctuations reveal simple exponential kinetics that reflect a small number of discrete states instead of the expected inhomogeneous dynamics. The have distribution of discrete dwell times, collected from an "ensemble" of 300 single molecules at each of a series of Mg(2+)and concentrations, fit well to a double exponential, which indicates that the RNA conformational changes can be described as a four-state molecules system. This finding is somewhat unexpected under [Mg(2+)] conditions in which this RNA does not achieve its native state. Observation of of discrete well-defined conformations in this large RNA that are stable on the seconds timescale at low [Mg(2+)](< .1 mM)concentrations, suggests that even at low ionic strength, with a tremendous number of possible (weak) interactions, a few critical interactions may number produce deep energy wells that allow for rapid averaging of motions within each well, and yield kinetics that are relatively which simple.

Single conformational molecule fluorescence resonance energy transfer (FRET) can be employed to study conformational heterogeneity and real-time dynamics of biological macromolecules. Here genomic we present single molecule studies on human genomic DNA G-quadruplex sequences that occur in the telomeres and in the promoter the of a proto-oncogene. The findings are discussed with respect to the proposed biological function(s) of such motifs in living cells.resonance

Ribonuclease occurring P is among the first ribozymes discovered, and is the only ubiquitously occurring ribozyme besides the ribosome. The bacterial RNase has P RNA is catalytically active without its protein subunit and has been studied for over two decades as a model a system for RNA catalysis, structure and folding. This review focuses on the thermodynamic, kinetic and structural frameworks derived from the among folding studies of bacterial RNase P RNA.

Enzymes there are complex macromolecules that catalyze chemical reactions at their active sites. Important information about catalytic interactions is commonly gathered by site perturbation or mutation of active site residues that directly contact substrates. However, active sites are engaged in intricate networks of that interactions within the overall structure of the macromolecule, and there is a growing body of evidence about the importance of Important peripheral interactions in the precise structural organization of the active site. Here, we use functional studies, in conjunction with published to structural information, to determine the effect of perturbation of a peripheral metal ion binding site on catalysis in a well-characterized hydroxyl catalytic RNA, the Tetrahymena thermophila group I ribozyme. We perturbed the metal ion binding site by site-specifically introducing a phosphorothioate reveal substitution in the ribozyme's backbone, replacing the native ligands (the pro-R(P) oxygen atoms at positions 307 and 308) with sulfur information, atoms. Our data reveal that these perturbations affect several reaction steps, including the chemical step, despite the absence of direct interactions contacts of this metal ion with the atoms involved in the chemical transformation. As structural probing with hydroxyl radicals did overall not reveal significant change in the three-dimensional structure upon phosphorothioate substitution, the effects are likely transmitted through local, rather subtle of conformational rearrangements. Addition of Cd(2+), a thiophilic metal ion, rescues some reaction steps but has deleterious effects on other steps.the These results suggest that native interactions in the active site may have been aligned by the naturally occurring peripheral residues ribozyme's and interactions to optimize the overall catalytic cycle.

Single-molecule their methods have matured into powerful and popular tools to probe the complex behaviour of biological molecules, due to their unique field abilities to probe molecular structure, dynamics and function, unhindered by the averaging inherent in ensemble experiments. This review presents an its overview of the burgeoning field of single-molecule biophysics, discussing key highlights and selected examples from its genesis to our projections into for its future. Following brief introductions to a few popular single-molecule fluorescence and manipulation methods, we discuss novel insights gained performed from single-molecule studies in key biological areas ranging from biological folding to experiments performed in vivo.

The for thermodynamics and kinetics for base-pair opening of the P1 duplex of the Tetrahymena group I ribozyme were studied by NMR The hydrogen exchange experiments. The apparent equilibrium constants for base pair opening were measured for most of the imino protons in pair the P1 duplex using the base catalysts NH(3), or TRIS. These equilibrium constants were also measured for several modified P1 the duplexes, and the C-2.G23 base pair was the most stable base pair in all the duplexes. The conserved U-1.G22 base catalytic pair is required for activity of the ribozyme and the data here show that this wobble base pair destabilizes neighboring base base pairs on only one side of the wobble. A 2'-OMe modification on the U-3 residue stabilized its own base base pair but had little effect on the neighboring base pairs. Three base pairs, U-1.G22, C-2.G23 and A2.U21 showed unusual equilibrium also constants for opening and possible implications of the opening thermodynamics of these base pairs on the undocking rates of the with P1 helix with catalytic core are discussed.

Many The technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio rejection (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a DNA thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the years background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background processing rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single-dye molecules. By labeling in a DNA molecule or a DNA/protein complex with a donor/acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used a to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the dramatically core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday data junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.

The into I-AniI maturase facilitates self-splicing of a mitochondrial group I intron in Aspergillus nidulans. Binding occurs in at least two steps:docking first, a specific but labile encounter complex rapidly forms and then this intermediate is slowly resolved into a native, catalytically important active RNA/protein complex. Here we probe the structure of the RNA throughout the assembly pathway. Although inherently unstable, the intron I core, when bound by I-AniI, undergoes rapid folding to a near-native state in the encounter complex. The next transition includes sequestered the slow destabilization and docking into the core of the peripheral stacked helix that contains the 5' splice site. Mutational site analyses confirm that both transitions are important for native complex formation. We propose that protein-driven destabilization and docking of the important peripheral stacked helix lead to subtle changes in the I-AniI binding site that facilitate native complex formation. These results support core, an allosteric-feedback mechanism of RNA-protein recognition in which proteins engaged in an intermediate complex can influence RNA structure far from get their binding sites. The linkage of these changes to stable binding ensures that the protein and RNA do not get intermediate sequestered in nonfunctional complexes.

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

Like vitro. many structured RNAs, the Tetrahymena group I ribozyme is prone to misfolding. Here we probe a long-lived misfolded species, referred misfolded to as M, and uncover paradoxical aspects of its structure and folding. Previous work indicated that a non-native local secondary to structure, termed alt P3, led to formation of M during folding in vitro. Surprisingly, hydroxyl radical footprinting, fluorescence measurements with Here site-specifically incorporated 2-aminopurine, and functional assays indicate that the native P3, not alt P3, is present in the M state.common The paradoxical behavior of alt P3 presumably arises because alt P3 biases folding toward M, but, after commitment to this structure folding pathway and before formation of M, alt P3 is replaced by P3. Further, structural and functional probes demonstrate that M the misfolded ribozyme contains extensive native structure, with only local differences between the two states, and the misfolded structure even presumably possesses partial catalytic activity. Despite the similarity of these structures, re-folding of M to the native state is very slow more and is strongly accelerated by urea, Na(+), and increased temperature and strongly impeded by Mg(2+) and the presence of native formation peripheral contacts. The paradoxical observations of extensive native structure within the misfolded species but slow conversion of this species to paradoxical the native state are readily reconciled by a model in which the misfolded state is a topological isomer of the is native state, and computational results support the feasibility of this model. We speculate that the complex topology of RNA secondary differences structures and the inherent rigidity of RNA helices render kinetic traps due to topological isomers considerably more common for RNA increased than for proteins.

The a GAAA tetraloop-receptor motif is a commonly occurring tertiary interaction in RNA. This motif usually occurs in combination with other tertiary important interactions in complex RNA structures. Thus, it is difficult to measure directly the contribution that a single GAAA tetraloop-receptor interaction coefficients makes to the folding properties of a RNA. To investigate the kinetics and thermodynamics for the isolated interaction, a GAAA This tetraloop domain and receptor domain were connected by a single-stranded A(7) linker. Fluorescence resonance energy transfer (FRET) experiments were used GAAA to probe intramolecular docking of the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where for the charge of the ion was the most important factor in determining the concentration of the ion required to promote Hill docking {[Co(NH(3))(6)(3+)]<<[Ca(2+)],[Mg(2+)],[Mn(2+)]<<[Na(+)],[K(+)]}. Analysis of metal ion cooperativity yielded Hill coefficients of approximately 2 experiments for Na(+)- or K(+)-dependent docking versus approximately 1 for the divalent ions and Co(NH(3))(6)(3+). Ensemble stopped-flow FRET kinetic measurements yielded on an apparent activation energy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U(7) and A(14) single-stranded linkers were to investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking. These studies showed that complex the single-stranded region functions primarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker was also K(+)-dependent investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking is discussed.

Depending between on the nucleotide sequence and the temperature and other conditions, RNA hairpin folding kinetics can be very complex. The complexity tests with a wide range of cooperative and noncooperative kinetic behaviors arises from the interplay between the formation of the loops,and the disruption of the misfolded states, and the formation of the rate-limiting base stacks. With a rate constant model and other a kinetic cluster theory, we explore the broad landscape for RNA hairpin folding kinetics. The model is validated through direct rate tests against several experimental measurements. The general folding kinetic mechanisms and the predicted great variety of folding kinetics are directly a applicable and quantitatively testable in experiments. The results from the present study suggest that (1) previous experimental findings based on and the individual hairpins revealed only a small fraction of much broader and more complex RNA hairpin folding landscapes,(2) even constant for structures as simple as hairpins, universal folding time scale and pathways do not exist, and (3) to treat the folding loop size as the sole factor to determine the hairpin folding rate is an oversimplification.

Foldable with polymers with alternating single-strand deoxyribonucleic acid (ssDNA) and planar fluorescent organic chromophores can self-organize into folded nanostructures and hence are that hybrid foldamers with biological sequences and synthetic properties. The biological sequence provides highly specific molecular recognition properties, while the physical controlled properties of synthetic chromophores offer sensitive fluorescence detection. In this paper, we describe that rational designed hybrid foldamers exhibit potential deoxyribonucleic in the detection of polynucleotides. Under strictly controlled laboratory conditions, fluorescence measurements indicate that configuration change due to binding of as polynucleotides with one or two mismatched bases can be readily distinguished. These results shed light on the design and construction one of nanostructured foldamers with actuator and sensory properties, which may find important applications as biological probes.

Single-molecule Recent experiments significantly expand our capability to characterize complex dynamics of biological processes. This relatively new approach has contributed significantly to and our understanding of the RNA folding problem. Recent single-molecule experiments, together with structural and biochemical characterizations of RNA at the secondary ensemble level, show that RNA molecules typically fold across a highly rugged energy landscape. As a result, long-lived folding intermediates,characterize multiple folding pathways, and heterogeneous conformational dynamics are commonly found for RNA enzymes. While initial results have suggested that stable provide secondary structures are partly responsible for the rugged energy landscape of RNA, a complete mechanistic understanding of the complex folding folding behavior has not yet been obtained. A combination of single-molecule experiments, which are well suited to analyze transient and heterogeneous secondary dynamic behaviors, with ensemble characterizations that can provide structural information at a superior resolution will likely provide more answers.