Single-molecule FRET reveals nucleotide-driven conformational changes in molecular machines and their link to RNA unwinding and DNA supercoiling

Many complex cellular processes in the cell are catalysed at the expense of ATP hydrolysis. The enzymes involved bind and hydrolyse ATP and couple ATP hydrolysis to the catalysed process via cycles of nucleotide-driven conformational changes. In this review, I illustrate how smFRET (single-molecule fluorescence resonance energy transfer) can define the underlying conformational changes that drive ATP-dependent molecular machines. The first example is a DEAD-box helicase that alternates between two different conformations in its catalytic cycle during RNA unwinding, and the second is DNA gyrase, a topoisomerase that undergoes a set of concerted conformational changes during negative supercoiling of DNA.     

Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes

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Biophysical analysis of influenza A virus RNA promoter at physiological temperatures

Each segment of the influenza A virus (IAV) genome contains conserved sequences at the 5'- and 3'-terminal ends, which form the promoter region necessary for polymerase binding and initiation of RNA synthesis. Although several models of interaction have been proposed it remains unclear if these two short, partially complementary, and highly conserved sequences can form a stable RNA duplex at physiological temperatures. First, our time-resolved FRET analysis revealed that a 14-mer 3'-RNA and a 15-mer 5'-RNA associate in solution, even at 42 °C. We also found that a nonfunctional RNA promoter containing the 3'-G3U mutation, as well as a promoter containing the compensatory 3'-G3U/C8A mutations, was able to form a duplex as efficiently as wild type. Second, UV melting analysis demonstrated that the wild-type and mutant RNA duplexes have similar stabilities in solution. We also observed an increase in thermostability for a looped promoter structure. The absence of differences in the stability and binding kinetics between wild type and a nonfunctional sequence suggests that the IAV promoter can be functionally inactivated without losing the capability to form a stable RNA duplex. Finally, using uridine specific chemical probing combined with mass spectrometry, we confirmed that the 5' and 3' sequences form a duplex which protects both RNAs from chemical modification, consistent with the previously published panhandle structure. These data support that these short, conserved promoter sequences form a stable complex at physiological temperatures, and this complex likely is important for polymerase recognition and viral replication.     

Mutational analysis of the promoter required for influenza virus virion RNA synthesis.

An in vitro RNA synthesis system was established in which the influenza virus virion (minus-sense) RNA was made from the synthetic plus-sense RNA (cRNA) template by the purified viral polymerase complex. The cRNA promoter was studied by mutational analysis using the in vitro system, and on the basis of these experiments, the first 11 nucleotides of the 3' noncoding sequence were found to contain the minimum promoter required for virion RNA synthesis. The addition of extra nucleotides at the 3' end decreased the promoter activity of the templates, indicating that the viral polymerase does not recognize an internal promoter efficiently. The wild-type and mutated RNA templates were also tested in vivo by using the ribonucleoprotein transfection system. In contrast to the in vitro system, it was found that the majority of mutations at the 3'-terminal sequence significantly decreased or abolished chloramphenicol acetyltransferase (CAT) expression. These results suggest that the cRNA promoter overlaps other essential cis elements required for chloramphenicol acetyltransferase expression in vivo.     

Single-molecule FRET study of SNARE-mediated membrane fusion

Membrane fusion is one of the most important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. Proteins, called SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor), play a central role in the fusion process that is also regulated by several accessory proteins. In order to study the SNARE-mediated membrane fusion, the in vitro protein reconstitution assay involving ensemble FRET (fluorescence resonance energy transfer) has been used over a decade. In this mini-review, we describe several single-molecule-based FRET approaches that have been applied to this field to overcome the shortage of the bulk assay in terms of protein and fusion dynamics.     

Single-molecule FRET of protein structure and dynamics - a primer

Single-molecule spectroscopy has developed into a widely used method for probing the structure, dynamics, and mechanisms of biomolecular systems, especially in combination with Förster resonance energy transfer (FRET). In this introductory tutorial, essential concepts and methods will be outlined, from the FRET process and the basic considerations for sample preparation and instrumentation to some key elements of data analysis and photon statistics. Different approaches for obtaining dynamic information over a wide range of timescales will be explained and illustrated with examples, including the quantitative analysis of FRET efficiency histograms, correlation spectroscopy, fluorescence trajectories, and microfluidic mixing.     

RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase in human cells reveals requirements for de novo initiation and protein-protein interaction

Brome mosaic virus (BMV) is a model positive-strand RNA virus whose replication has been studied in a number of surrogate hosts. In transiently transfected human cells, the BMV polymerase 2a activated signaling by the innate immune receptor RIG-I, which recognizes de novo-initiated non-self-RNAs. Active-site mutations in 2a abolished RIG-I activation, and coexpression of the BMV 1a protein stimulated 2a activity. Mutations previously shown to abolish 1a and 2a interaction prevented the 1a-dependent enhancement of 2a activity. New insights into 1a-2a interaction include the findings that helicase active site of 1a is required to enhance 2a polymerase activity and that negatively charged amino acid residues between positions 110 and 120 of 2a contribute to interaction with the 1a helicase-like domain but not to the intrinsic polymerase activity. Confocal fluorescence microscopy revealed that the BMV 1a and 2a colocalized to perinuclear region in human cells. However, no perinuclear spherule-like structures were detected in human cells by immunoelectron microscopy. Sequencing of the RNAs coimmunoprecipitated with RIG-I revealed that the 2a-synthesized short RNAs are derived from the message used to translate 2a. That is, 2a exhibits a strong cis preference for BMV RNA2. Strikingly, the 2a RNA products had initiation sequences (5'-GUAAA-3') identical to those from the 5' sequence of the BMV genomic RNA2 and RNA3. These results show that the BMV 2a polymerase does not require other BMV proteins to initiate RNA synthesis but that the 1a helicase domain, and likely helicase activity, can affect RNA synthesis by 2a.     

FRET analysis reveals distinct conformations of IN tetramers in the presence of viral DNA or LEDGF/p75

A tetramer of HIV-1 integrase (IN) stably associates with the viral DNA ends to form a fully functional concerted integration intermediate. LEDGF/p75, a key cellular binding partner of the lentiviral enzyme, also stabilizes a tetrameric form of IN. However, functional assays have indicated the importance of the order of viral DNA and LEDGF/p75 addition to IN for productive concerted integration. Here, we employed Förster Resonance Energy Transfer (FRET) to monitor assembly of individual IN subunits into tetramers in the presence of viral DNA and LEDGF/p75. The IN–viral DNA and IN–LEDGF/p75 complexes yielded significantly different FRET values suggesting two distinct IN conformations in these complexes. Furthermore, the order of addition experiments indicated that FRET for the preformed IN–viral DNA complex remained unchanged upon its subsequent binding to LEDGF/p75, whereas pre-incubation of LEDGF/p75 and IN followed by addition of viral DNA yielded FRET very similar to the IN–LEDGF/p75 complex. These findings provide new insights into the structural organization of IN subunits in functional concerted integration intermediates and suggest that differential multimerization of IN in the presence of various ligands could be exploited as a plausible therapeutic target for development of allosteric inhibitors.     

Solution structure of the influenza A virus cRNA promoter: implications for differential recognition of viral promoter structures by RNA-dependent RNA polymerase

Influenza A virus replication requires the interaction of viral RNA-dependent RNA polymerase (RdRp) with promoters in both the RNA genome (vRNA) and the full-length complementary RNA (cRNA) which serve as templates for the generation of new vRNAs. Although RdRp binds both promoters effectively, it must also discriminate between them because they serve different functional roles in the viral life cycle. Even though the inherent asymmetry between two RNA promoters is considered as a cause of the differential recognition by the RdRp, the structural basis for the ability of the RdRp to recognize the RNA promoters and discriminate effectively between them remains unsolved. Here we report the structure of the cRNA promoter of influenza A virus as determined by heteronuclear magnetic resonance spectroscopy. The terminal region is extremely unstable and does not have a rigid structure. The major groove of the internal loop is widened by the displacement of a novel A*(UU) motif toward the minor groove. These internal loop residues show distinguishable dynamic characters, with differing motional timescales for each residue. Comparison of the cRNA promoter structure with that of the vRNA promoter reveals common structural and dynamic elements in the internal loop, but also differences that provide insight into how the viral RdRp differentially recognizes the cRNA and vRNA promoters.     

Mechanistic aspects of promoter binding and chain initiation by RNA polymerase

Summary The physicochemical properties of the interactions of RNA polymerase (RPase) with promoter and nonspecific DNA sequences have been investigated. These show that nonspecific binding is principally an ionic interaction and that promoter binding is more complex, involving nonionic interactions. Nonspecific binding has been shown to be very important in the promoter search, and one-dimensional diffusion can account for the rate at which RPase finds the promoter. Significant differences have been reported in the binding process for various promoters and in the effects of regulatory proteins. Further investigation of these differences will lead to a better understanding of the selectivity and regulation of the initiation process.The pathways of the initiation process have been outlined, by recent studies and considerable progress has been made in determining the rates of interconversion of the intermediate states. A number of questions remain about the detail of initiation and the effects of various parameters on the reactions. Of particular importance is the identification of the point at which the enzyme becomes truly processive. In addition, the step which is rate limiting has not been identified in either the productive or nonproductive process. The mechanistic features of the steps after bond formation are just beginning to yield to investigation.Use of substrate analogs with RPase has led to a picture of the polymerization site according to the ability of the enzyme to incorporate analogs. Base specificity appears to be determined primarily by interaction with the template rather than the enzyme, but the ribose moiety must interact with the site quite specifically. The orientation of the phosphate residues has been determined by NMR, which has also proved to be a valuable probe of the initiation site. At this site base specificity is resident in the enzyme and expressed through the interaction of the base and intrinsic metal, as shown by studies with the Cobalt substituted enzyme. In both initiation and polymerization, the reaction has been shown to proceed by inversion of configuration. Techniques similar to those used for initiation will probably be applied to the polymerization reaction as well, which has not recently received as much attention with respect to mechanism. Functional phenomena such as pausing make the polymerization process particularly promising for producing insight into RPase reactions.     

Single-molecule FRET study of denaturant induced unfolding of RNase H

Single-molecule fluorescence (F?rster) resonance energy transfer (FRET) experiments were performed on surface-immobilized RNase H molecules as a function of the concentration of the chemical denaturant guanidinium chloride (GdmCl). For comparison, we measured ensemble FRET on RNase H solutions. The single-molecule approach allowed us to study FRET distributions of the subpopulation of unfolded molecules without interference from the folded population. The unfolded ensemble experienced a continuous shift of the FRET efficiency distribution with increasing concentration of GdmCl, indicating a heterogeneous population of expanding, unfolded polypeptide chains. We have analyzed the behavior of the unfolded state quantitatively with a model in which the unfolded state is described by a continuum of substates, with the free energy of each substate linearly coupled to its m-value, the proportionality coefficient between free energy and denaturant activity. By fitting this model to the data, we have derived energetic and structural parameters that describe the unfolded state ensemble. Specifically, we have found that the average size of the unfolded state increases from 23-38 A between 0 and 6 M denaturant. Excellent agreement was achieved between the fitted model and our FRET measurements, and with previously published nuclear magnetic resonance and small-angle X-ray scattering data.     

RNA polymerase of influenza virus. III. Isolation of RNA polymerase-RNA complexes from influenza virus PR8

Ribonucleoprotein (RNP) cores with RNA-synthesizing activity were prepared in two fractions, M protein-free and M protein-associated, from detergent-treated influenza virus PR8 by centrifugation through a discontinuous triple gradient of cesium sulfate, glycerol, and NP-40. The M-free RNP was fractionated by phosphocellulose column chromatography into two major RNP forms, A and B, which differed in the content of P proteins, while the M-associated RNP gave only the low P-content Form-B RNP. Starting from the high P-content Form-A RNP, an RNA-P proteins complex virtually free from NP protein was isolated by cesium sulfate equilibrium centrifugation. The complex, containing only three P proteins (P1, P2, and P3), was still active in catalyzing RNA synthesis in vitro without addition of exogenous template, indicating that NP protein is not required for the catalysis of RNA synthesis. RNA synthesis by the isolated RNA-P proteins complex was dependent on either ApG or capped RNA primers, and required four ribonucleoside triphosphates as substrates. The RNA product in this reaction was hybridizable to viral RNA. A complex of one each of the three P proteins was separated from RNA by glycerol gradient centrifugation after ribonuclease treatment or cesium chloride equilibrium centrifugation.     

Single-molecule investigations of RNA dissociation

Given the essential cellular roles for ribonucleic acids (RNAs) it is important to understand the stability of three-dimensional structures formed by these molecules. This study aims to investigate the dissociation energy landscape for simple RNA structures via atomic-force-microscopy-based single-molecule force-spectroscopy measurements. This approach provides details on the locations and relative heights of the energy barriers to dissociation, and thus information upon the relative kinetic stabilities of the formed complexes. Our results indicate that a simple dodecamer RNA helix undergoes a forced dissociation process similar to that previously observed for DNA oligonucleotides. Incorporating a UCU bulge motif is found to introduce an additional energy barrier closer to the bound state, and also to destabilize the duplex. In the absence of magnesium ions a duplex containing this UCU bulge is destabilized and a single, shorter duplex is formed. These results reveal that a bulge motif impacts upon the forced dissociation of RNA and produces an energy landscape sensitive to the presence of magnesium ions. Interestingly, the obtained data compare well with previously reported ensemble measurements, illustrating the potential of this approach to improve our understanding of RNA stability and dissociation kinetics.