Eitan Lerner

Multiple conformations of full-length p53 detected with single-molecule fluorescence resonance energy transfer

The tumor suppressor p53 is a member of the emerging class of proteins that have both folded and intrinsically disordered domains, which are a challenge to structural biology. Its N-terminal domain (NTD) is linked to a folded core domain, which has a disordered link to the folded tetramerization domain, which is followed by a disordered C-terminal domain. The quaternary structure of human p53 has been solved by a combination of NMR spectroscopy, electron microscopy, and small-angle X-ray scattering (SAXS), and the NTD ensemble structure has been solved by NMR and SAXS. The murine p53 is reported to have a different quaternary structure, with the N and C termini interacting. Here, we used single-molecule FRET (SM-FRET) and ensemble FRET to investigate the conformational dynamics of the NTD of p53 in isolation and in the context of tetrameric full-length p53 (flp53). Our results showed that the isolated NTD was extended in solution with a strong preference for residues 66–86 forming a polyproline II conformation. The NTD associated weakly with the DNA binding domain of p53, but not the C termini. We detected multiple conformations in flp53 that were likely to result from the interactions of NTD with the DNA binding domain of each monomeric p53. Overall, the SM-FRET results, in addition to corroborating the previous ensemble findings, enabled the identification of the existence of multiple conformations of p53, which are often averaged and neglected in conventional ensemble techniques. Our study exemplifies the usefulness of SM-FRET in exploring the dynamic landscape of multimeric proteins that contain regions of unstructured domains.

Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase

Significance Transcription initiation by RNA polymerase (RNAP) is a highly regulated rate-limiting step in many genes and involves numerous intermediate states that remain incompletely understood. Here, we report the characterization of a previously hypothesized slow initiation pathway involving RNAP backtracking and pausing. This backtracked and paused state is observed when all nucleoside triphosphates (NTPs) are present at physiologically relevant concentrations, but becomes more prevalent with unbalanced NTP levels, which may occur in vivo under conditions of metabolic stress. Pausing and backtracking in initiation may play an important role in regulating RNAP transcription. Moreover, similar RNA backtracked states may contribute to promoter-proximal pausing among eukaryotic RNA polymerase II enzymes. Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2- or 4-base transcript). In addition, the RNAP–GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused–backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.

FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing Single-Molecule FRET

Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores. While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by software implementation. In particular, despite the widespread application of smFRET, no complete software package for smFRET burst analysis is freely available to date. In this paper, we introduce FRETBursts, an open source software for analysis of freely-diffusing smFRET data. FRETBursts allows executing all the fundamental steps of smFRET bursts analysis using state-of-the-art as well as novel techniques, while providing an open, robust and well-documented implementation. Therefore, FRETBursts represents an ideal platform for comparison and development of new methods in burst analysis. We employ modern software engineering principles in order to minimize bugs and facilitate long-term maintainability. Furthermore, we place a strong focus on reproducibility by relying on Jupyter notebooks for FRETBursts execution. Notebooks are executable documents capturing all the steps of the analysis (including data files, input parameters, and results) and can be easily shared to replicate complete smFRET analyzes. Notebooks allow beginners to execute complex workflows and advanced users to customize the analysis for their own needs. By bundling analysis description, code and results in a single document, FRETBursts allows to seamless share analysis workflows and results, encourages reproducibility and facilitates collaboration among researchers in the single-molecule community.

Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (via PIFE) and energy transfer efficiency (via FRET) can simultaneously report on e.g., the conformational state of double stranded DNA (dsDNA) following its interaction with unlabelled proteins (BamHI, EcoRV, and T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules. Finally, we study transcription initiation and scrunching of E. coli RNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble.

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

Watching single molecules in motion Structural techniques such as x-ray crystallography and electron microscopy give insight into how macromolecules function by providing snapshots of different conformational states. Function also depends on the path between those states, but to see that path involves watching single molecules move. This became possible with the advent of single-molecule Förster resonance energy transfer (smFRET), which was first implemented in 1996. Lerner et al. review how smFRET has been used to study macromolecules in action, providing mechanistic insights into processes such as DNA repair, transcription, and translation. They also describe current limitations of the approach and suggest how future developments may expand the applications of smFRET. Science, this issue p. eaan1133 BACKGROUND Biomolecular mechanisms are typically inferred from static structural “snapshots” obtained by x-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo–electron microscopy (cryo-EM). In these approaches, mechanisms have to be validated using additional information from established biochemical and biophysical assays. However, linking conformational states to biochemical function requires the ability to resolve structural dynamics, as macromolecular structure can be intrinsically dynamic or altered upon ligand binding. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying such structural dynamics under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments both confirmed previous hypotheses and discovered new fundamental biological mechanisms relevant for DNA maintenance, replication and transcription, translation, protein folding, enzymatic function, and membrane transport. We review the evolution of smFRET as a key tool for “dynamic structural biology” over the past 22 years and highlight the prospects for its use in applications such as biosensing, high-throughput screening, and molecular diagnostics. ADVANCES FRET was first identified in the 1920s by Cario, Franck, and Perrin. In the late 1940s, Förster and Oppenheimer independently formulated a quantitative theory of the energy transfer between a pair of point dipoles. Stryer and Haugland verified this theory in the late 1960s and coined the term “spectroscopic ruler” for FRET. Simultaneously, Hirschfeld, and later Moerner and Orrit, pioneered optical single-molecule detection methods leading to the first demonstration of smFRET in 1996. This breakthrough made it possible to study heterogeneous systems, dynamic processes, and transient conformational changes on the nanometer scale. The smFRET technique was rapidly adopted by various research groups to provide mechanistic answers in diverse areas of biological research. In early pioneering applications of smFRET in biochemistry, Ha et al. visualized the conformational dynamics of the staphylococcal nuclease enzyme; Deniz et al. obtained information on the structural dynamics of double-stranded DNA; and Zhuang et al. studied the conformation of individual RNA enzyme molecules and their folding dynamics in equilibrium. These pioneering studies were followed by others that used smFRET to unravel the inner workings of helicases and topoisomerases, DNA replication, DNA repair, transcription, translation, enzymatic reactions, molecular motors, membrane proteins, nucleic acids, protein and RNA folding, ribozyme catalysis, and many other molecular mechanisms. OUTLOOK During the past two decades, smFRET has grown into a mature toolset with capabilities to explore dynamic structural biology for both equilibrium and non-equilibrium reactions. The one-dimensional (“ruler”) character of the FRET approach, however, only captures the complex three-dimensional structure of a system and needs to be complemented by other techniques that can provide additional information about the respective biochemical states of macromolecules. Approaches that explore smFRET combinations with other biophysical techniques (patch-clamp, optical, and magnetic tweezers; atomic force microscopy; microfluidics) or photophysical effects are hence gaining attention. Although smFRET is particularly useful for the observation of dynamic conformational changes and subpopulations, FRET efficiencies also carry very precise information on the actual distance between fluorophores attached to distinct moieties of a macromolecule. As shown by recent work from many laboratories (such as those of Seidel, Michaelis, Hugel, and Grubmüller), this quantitative information can be used to help define biological structures and in the future should find a place in the protein database of molecular structures. smFRET has so far mostly been used for in vitro experiments but can be used additionally to monitor conformational dynamics and heterogeneity in live cells. “In vivo smFRET” has recently emerged as a promising methodology, demonstrated by the groups of Sakon, Weninger, Schuler, and Kapanidis among others. We envision that further technological developments will expand smFRET applications beyond dynamic structural biology to allow fast nonequilibrium kinetic studies, high-throughput drug screening, and molecular diagnostics. Advancements of these applications will be impactful for systems that are highly heterogeneous and dynamic. Dynamic structural biology using smFRET. Left: Principle of FRET as a molecular ruler. In a system with a pair of dyes, after the donor dye (D) is excited, it transfers the excitation energy to a nearby acceptor dye (A; top) with an efficiency (E) that depends on the sixth power of the distance between the dyes (bottom). Right: Use of FRET to study structural dynamics at the single-macromolecule level. The experimental setup (top), a combination of single-molecule fluorescence microscopy and spectroscopy, can be used to determine conformational states or dynamics in solution or on immobilized molecules. Here E is calculated per each single-molecule burst of photons, and bursts (n) are accumulated in E histograms (middle) or for different time bins to form a single-molecule E trajectory (bottom). Classical structural biology can only provide static snapshots of biomacromolecules. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying dynamics in macromolecular structures under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments have confirmed previously hypothesized mechanisms and provided new insights into many fundamental biological processes, such as DNA maintenance and repair, transcription, translation, and membrane transport. We review 22 years of contributions of smFRET to our understanding of basic mechanisms in biochemistry, molecular biology, and structural biology. Additionally, building on current state-of-the-art implementations of smFRET, we highlight possible future directions for smFRET in applications such as biosensing, high-throughput screening, and molecular diagnostics.

A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement–Förster-Type Resonance Energy Transfer (PIFE-FRET)

Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90° intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data, and show how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein–DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems.

Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

Different types of pausing modes during transcription initiation

ABSTRACT In many cases, initiation is rate limiting to transcription. This due in part to the multiple cycles of abortive transcription that delay promoter escape and the transition from initiation to elongation. Pausing of transcription in initiation can further delay promoter escape. The previously hypothesized pausing in initiation was confirmed by two recent studies from Duchi et al.1 and from Lerner, Chung et al.2 In both studies, pausing is attributed to a lack of forward translocation of the nascent transcript during initiation. However, the two works report on different pausing mechanisms. Duchi et al. report on pausing that occurs during initiation predominantly on-pathway of transcript synthesis. Lerner, Chung et al. report on pausing during initiation as a result of RNAP backtracking, which is off-pathway to transcript synthesis. Here, we discuss these studies, together with additional experimental results from single-molecule FRET focusing on a specific distance within the transcription bubble. We show that the results of these studies are complementary to each other and are consistent with a model involving two types of pauses in initiation: a short-lived pause that occurs in the translocation of a 6-mer nascent transcript and a long-lived pause that occurs as a result of 1–2 nucleotide backtracking of a 7-mer transcript.

Studying transcription initiation by RNA polymerase with diffusion-based single-molecule fluorescence

Over the past decade, fluorescence‐based single‐molecule studies significantly contributed to characterizing the mechanism of RNA polymerase at different steps in transcription, especially in transcription initiation. Transcription by bacterial DNA‐dependent RNA polymerase is a multistep process that uses genomic DNA to synthesize complementary RNA molecules. Transcription initiation is a highly regulated step in E. coli, but it has been challenging to study its mechanism because of its stochasticity and complexity. In this review, we describe how single‐molecule approaches have contributed to our understanding of transcription and have uncovered mechanistic details that were not observed in conventional assays because of ensemble averaging.

FRETBursts: Open Source Burst Analysis Toolkit for Confocal Single-Molecule FRET

Single-molecule Forster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool in studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume it emits a burst of photons that can be detected by single-photon avalanche detectors (SPADs). The intensities of donor and acceptor fluorescence can then be related to the distance between the two dyes. The analysis of smFRET experiments involves identifying photon bursts from single-molecules in a continuous stream of photon, estimating the background and other correction factors, filtering and finally extracting the corrected FRET efficiencies for each sub-population in the sample. In this paper we introduce FRETBursts, a software for confocal smFRET data analysis which allows executing a complete burst analysis pipeline by using state-of-the-art algorithms. FRETBursts is an open source python package that we envision both as toolkit for research and new developments in burst analysis and as reference implementation of commonly employed algorithms. We follow the highest standard in software development to ensure that the source is easy to read, well documented and thoroughly tested. Moreover, in an effort to lower the barriers to computational reproducibility, we embrace a modern workflow based on Jupyter notebooks that allows to capture of the whole process from raw data to figures within a single document.Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3–10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores. While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by so.ware implementation. In particular, despite the widespread application of smFRET, no complete so.ware package for smFRET burst analysis is freely available to date. In this paper, we introduce FRETBursts, an open source software for analysis of freely-diffusing smFRET data. FRETBursts allows executing all the fundamental steps of smFRET bursts analysis using state-of-the-art as well as novel techniques, while providing an open, robust and welldocumented implementation. Therefore, FRETBursts represents an ideal platform for comparison and development of new methods in burst analysis. We employ modern software engineering principles in order to minimize bugs and facilitate long-term maintainability. Furthermore, we place a strong focus on reproducibility by relying on Jupyter notebooks for FRETBursts execution. Notebooks are executable documents capturing all the steps of the analysis (including data files, input parameters, and results) and can be easily shared to replicate complete smFRET analyzes. Notebooks allow beginners to execute complex workflows and advanced users to customize the analysis for their own needs. By bundling analysis description, code and results in a single document, FRETBursts allows to seamless share analysis workflows and results, encourages reproducibility and facilitates collaboration among researchers in the single-molecule community.