Single-molecule Fluorescence Assays Research Articles

Dynamics of release factor recycling during translation termination in bacteria.

In bacteria, release of newly synthesized proteins from ribosomes during translation termination is catalyzed by class-I release factors (RFs) RF1 or RF2, reading UAA and UAG or UAA and UGA codons, respectively. Class-I RFs are recycled from the post-termination ribosome by a class-II RF, the GTPase RF3, which accelerates ribosome intersubunit rotation and class-I RF dissociation. How conformational states of the ribosome are coupled to the binding and dissociation of the RFs remains unclear and the importance of ribosome-catalyzed guanine nucleotide exchange on RF3 for RF3 recycling in vivo has been disputed. Here, we profile these molecular events using a single-molecule fluorescence assay to clarify the timings of RF3 binding and ribosome intersubunit rotation that trigger class-I RF dissociation, GTP hydrolysis, and RF3 dissociation. These findings in conjunction with quantitative modeling of intracellular termination flows reveal rapid ribosome-dependent guanine nucleotide exchange to be crucial for RF3 action in vivo.

Single-molecule fluorescence assays for studying the mechanism of DNA cleavage by CRISPR-Cas12a

Single-molecule fluorescence assays for studying the mechanism of DNA cleavage by CRISPR-Cas12a

Three kinetically distinct but compatible routes of Rho-dependent termination uncovered by single-molecule transcription imaging.

When gene transcription for RNA synthesis terminates in a factor-dependent manner, Rho is a general termination factor in bacteria, but its mechanism has been elusive in many aspects. Various models have been proposed for the pre-terminational interaction of Rho with RNA polymerase (RNAP), for the terminational release of RNA transcript, and for the post-terminational outcome. We developed several single-molecule fluorescence assays in order to monitor those three steps of termination mediated by Escherichia coli Rho. We find that the different mechanisms previously proposed for each step co-exist compatibly together in any single terminator but three kinetically distinct routes take place with each terminator. Rho mostly binds nascent RNA first at a rut site, translocates on RNA and catches up with the paused RNAP at the termination site. The fastest route is that this catch-up Rho first mediates RNA shearing for RNA-only release and consequently one-dimensional recycling of the DNA-bound RNAP. The catch-up Rho later mediates RNAP displacement in the next but the most frequent route to decompose RNAP·DNA·RNA complexes in a step for three-dimensional recycling of the dissociated RNAP. Contrastingly, a minor fraction of Rho pre-binds RNAP prior to the terminational pausing site and stands by for an incipient RNA rut site. This stand-by Rho lastly mediates only RNAP displacement for the decomposing outcome in the slowest route, despite interacting with RNAP earlier than the catch-up Rho. Furthermore, we uncover that transcriptional pause extension benefits only the stand-by Rho's termination, and the essential pause does not need to be long for the catch-up Rho's terminations The riboswitch-associated termination is controlled mainly by modulation of the stand-by. Thus, the three compatible routes of Rho-dependent termination not only operate on distinct timescales but also play different regulatory roles.

Spatiotemporally controlled generation of NTPs for single-molecule studies.

Many essential processes in the cell depend on proteins that use nucleoside triphosphates (NTPs). Methods that directly monitor the often-complex dynamics of these proteins at the single-molecule level have helped to uncover their mechanisms of action. However, the measurement throughput is typically limited for NTP-utilizing reactions, and the quantitative dissection of complex dynamics over multiple sequential turnovers remains challenging. Here we present a method for controlling NTP-driven reactions in single-molecule experiments via the local generation of NTPs (LAGOON) that markedly increases the measurement throughput and enables single-turnover observations. We demonstrate the effectiveness of LAGOON in single-molecule fluorescence and force spectroscopy assays by monitoring DNA unwinding, nucleosome sliding and RNA polymerase elongation. LAGOON can be readily integrated with many single-molecule techniques, and we anticipate that it will facilitate studies of a wide range of crucial NTP-driven processes.

Rho-dependent transcription termination proceeds via three routes

Rho is a general transcription termination factor in bacteria, but many aspects of its mechanism of action are unclear. Diverse models have been proposed for the initial interaction between the RNA polymerase (RNAP) and Rho (catch-up and stand-by pre-terminational models); for the terminational release of the RNA transcript (RNA shearing, RNAP hyper-translocation or displacing, and allosteric models); and for the post-terminational outcome (whether the RNAP dissociates or remains bound to the DNA). Here, we use single-molecule fluorescence assays to study those three steps in transcription termination mediated by E. coli Rho. We find that different mechanisms previously proposed for each step co-exist, but apparently occur on various timescales and tend to lead to specific outcomes. Our results indicate that three kinetically distinct routes take place: (1) the catch-up mode leads first to RNA shearing for RNAP recycling on DNA, and (2) later to RNAP displacement for decomposition of the transcriptional complex; (3) the last termination usually follows the stand-by mode with displacing for decomposing. This three-route model would help reconcile current controversies on the mechanisms.

Mg2+-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage.

CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)-distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg2+ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg2+-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.

Dynamic competition between a ligand and transcription factor NusA governs riboswitch-mediated transcription regulation

Cotranscriptional RNA folding is widely assumed to influence the timely control of gene expression, but our understanding remains limited. In bacteria, the fluoride (F-)-sensing riboswitch is a transcriptional control element essential to defend against toxic F- levels. Using this model riboswitch, we find that its ligand F- and essential bacterial transcription factor NusA compete to bind the cotranscriptionally folding RNA, opposing each other's modulation of downstream pausing and termination by RNA polymerase. Single-molecule fluorescence assays probing active transcription elongation complexes discover that NusA unexpectedly binds highly reversibly, frequently interrogating the complex for emerging, cotranscriptionally folding RNA duplexes. NusA thus fine-tunes the transcription rate in dependence of the ligand-responsive higher-order structure of the riboswitch. At the high NusA concentrations found intracellularly, this dynamic modulation is expected to lead to adaptive bacterial transcription regulation with fast response times.

How translation stops

Translation Protein synthesis concludes when a ribosome encounters a stop codon in a transcript, which triggers the recruitment of highly conserved release factors to liberate the protein product. Lawson et al. used traditional biochemical methods and single-molecule fluorescence assays to track the interplay of release factors with ribosomes and reveal the molecular choreography of termination. They identified two distinct classes of effectors, small molecules and mRNA sequences, that directly inhibited the release factors and promoted stop codon readthrough. These findings may buttress ongoing efforts to treat diseases caused by premature stop codons, which cause 11% of all heritable human diseases. Science , abi7801, this issue p. [876][1] [1]: /lookup/doi/10.1126/science.abi7801

Mechanisms that ensure speed and fidelity in eukaryotic translation termination.

Translation termination, which liberates a nascent polypeptide from the ribosome specifically at stop codons, must occur accurately and rapidly. We established single-molecule fluorescence assays to track the dynamics of ribosomes and two requisite release factors (eRF1 and eRF3) throughout termination using an in vitro-reconstituted yeast translation system. We found that the two eukaryotic release factors bound together to recognize stop codons rapidly and elicit termination through a tightly regulated, multistep process that resembles transfer RNA selection during translation elongation. Because the release factors are conserved from yeast to humans, the molecular events that underlie yeast translation termination are likely broadly fundamental to eukaryotic protein synthesis.

Stabilization of Hfq-mediated translational repression by the co-repressor Crc in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) govern translation of numerous transcripts during carbon catabolite repression. Here, Crc was shown to enhance Hfq-mediated translational repression of several mRNAs. We have developed a single-molecule fluorescence assay to quantitatively assess the cooperation of Hfq and Crc to form a repressive complex on a RNA, encompassing the translation initiation region and the proximal coding sequence of the P. aeruginosa amiE gene. The presence of Crc did not change the amiE RNA-Hfq interaction lifetimes, whereas it changed the equilibrium towards more stable repressive complexes. This observation is in accord with Cryo-EM analyses, which showed an increased compactness of the repressive Hfq/Crc/RNA assemblies. These biophysical studies revealed how Crc protein kinetically stabilizes Hfq/RNA complexes, and how the two proteins together fold a large segment of the mRNA into a more compact translationally repressive structure. In fact, the presence of Crc resulted in stronger translational repression in vitro and in a significantly reduced half-life of the target amiE mRNA in vivo. Although Hfq is well-known to act with small regulatory RNAs, this study shows how Hfq can collaborate with another protein to down-regulate translation of mRNAs that become targets for the degradative machinery.

Towards a Structural Mechanism for Sister Chromatid Cohesion Establishment at the Eukaryotic Replication Fork.

Cohesion between replicated chromosomes is essential for chromatin dynamics and equal segregation of duplicated genetic material. In the G1 phase, the ring-shaped cohesin complex is loaded onto duplex DNA, enriching at replication start sites, or "origins". During the same phase of the cell cycle, and also at the origin sites, two MCM helicases are loaded as symmetric double hexamers around duplex DNA. During the S phase, and through the action of replication factors, cohesin switches from encircling one parental duplex DNA to topologically enclosing the two duplicated DNA filaments, which are known as sister chromatids. Despite its vital importance, the structural mechanism leading to sister chromatid cohesion establishment at the replication fork is mostly elusive. Here we review the current understanding of the molecular interactions between the replication machinery and cohesin, which support sister chromatid cohesion establishment and cohesin function. In particular, we discuss how cryo-EM is shedding light on the mechanisms of DNA replication and cohesin loading processes. We further expound how frontier cryo-EM approaches, combined with biochemistry and single-molecule fluorescence assays, can lead to understanding the molecular basis of sister chromatid cohesion establishment at the replication fork.

Probing Structural Dynamics of the Closed-Loop Model in Translation Initiation

During translation initiation, communication between the mRNA 5′ and 3′ ends takes on a pivotal role for gene expression regulation. Binding of eukaryotic initiation factor 4F (eIF4F) at the 5′ cap, and of poly(A)-binding protein (Pab1p in yeast) to the 3′ poly(A) tail, is thought to bring the mRNA ends close together, due to a physical eIF4F-Pab1p interaction. Detailed studies have denoted that this “closed loop” arrangement has important functional consequences, including enhanced mRNA stability and synergistic enhancement of translation. A combination of biochemical, genetic, and structural studies from the past four decades have delineated the roles of each closed-loop factor; however, our understanding of the molecular architecture of this multiprotein-mRNA complex remains mostly limited to static snapshots. in addition, little is known on how this architecture is dynamically leveraged to promote and regulate translation initiation. To address this problem, we have developed single-molecule fluorescence assays to visualize when closed-loop factors are bound to the mRNA, while simultaneously probing mRNA end-to-end proximity (when mRNA ends are within ∼ 7 nm of each other). We observed the interaction of recombinant, fluorescently-labeled S. cerevisiae eIF4E and full-length mRNAs fluorescently labeled at their 3′ ends. Our results indicate that mRNA ends are intrinsically close in the absence of factors in vitro, and that a dominant role of Pab1p is to enhance eIF4F-mRNA association kinetics. We are applying this approach to understand the role of mRNA sequence, length, and secondary structure, and our data will provide new insights into the role of the closed loop in translational control.

Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1-40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.

Mechanistic studies of non-canonical amino acid mutagenesis.

Over the past decade, harnessing the cellular protein synthesis machinery to incorporate non-canonical amino acids (ncAAs) into tailor-made peptides has significantly advanced many aspects of molecular science. More recently, groundbreaking progress in our ability to engineer this machinery for improved ncAA incorporation has led to significant enhancements of this powerful tool for biology and chemistry. By revealing the molecular basis for the poor or improved incorporation of ncAAs, mechanistic studies of ncAA incorporation by the protein synthesis machinery have tremendous potential for informing and directing such engineering efforts. In this chapter, we describe a set of complementary biochemical and single-molecule fluorescence assays that we have adapted for mechanistic studies of ncAA incorporation. Collectively, these assays provide data that can guide engineering of the protein synthesis machinery to expand the range of ncAAs that can be incorporated into peptides and increase the efficiency with which they can be incorporated, thereby enabling the full potential of ncAA mutagenesis technology to be realized.

Single-Molecule Fluorescence Methods to Study Protein Exchange Kinetics in Supramolecular Complexes.

Recent single-molecule studies have demonstrated that the composition of multi-protein complexes can strike a balance between stability and dynamics. Proteins can dynamically exchange in and out of the complex depending on their concentration in solution. These exchange dynamics are a key determinant of the molecular pathways available to multi-protein complexes. It is therefore important that we develop robust and reproducible assays to study protein exchange. Using DNA replication as an example, we describe three single-molecule fluorescence assays used to study protein exchange dynamics. In the chase exchange assay, fluorescently labeled proteins are challenged by unlabeled proteins, where exchangeresults in the disappearance of the fluorescence signal. In the FRAP exchange assay, fluorescently labeled proteins are photobleached before exchange is measured by an increase in fluorescence as non-bleached proteins exchange into the complex. Finally, in the two-color exchange assay, proteins are labeled with two different fluorophores and exchange is visualized by detecting changes in color. All three assays compliment in their ability toelucidate the dynamic behavior of proteins in large biological systems.

KCNE1 is an auxiliary subunit of two distinct ion channel superfamilies

Determination of what is the specificity of subunits composing a protein complex is essential when studying gene variants on human pathophysiology. The pore-forming α-subunit KCNQ1, which belongs to the voltage-gated ion channel superfamily, associates to its β-auxiliary subunit KCNE1 to generate the slow cardiac potassium IKs current, whose dysfunction leads to cardiac arrhythmia. Using pharmacology, gene invalidation, and single-molecule fluorescence assays, we found that KCNE1 fulfils all criteria of a bona fide auxiliary subunit of the TMEM16A chloride channel, which belongs to the anoctamin superfamily. Strikingly, assembly with KCNE1 switches TMEM16A from a calcium-dependent to a voltage-dependent ion channel. Importantly, clinically relevant inherited mutations within the TMEM16A-regulating domain of KCNE1 abolish the TMEM16A modulation, suggesting that the TMEM16A-KCNE1 current may contribute to inherited pathologies. Altogether, these findings challenge the dogma of the specificity of auxiliary subunits regarding protein complexes and questions ion channel classification.

Heterogeneous Dynamics of Protein-RNA Interactions across Transcriptome-Derived Messenger RNA Populations.

Dynamic RNA-protein interactions underpin numerous molecular control mechanisms in biology. However, relatively little is known about the kinetic landscape of protein interactions with full-length RNAs. The extent to which interaction kinetics vary for the same RNA element across the transcriptome and the molecular determinants of variability therefore remain poorly defined. Moreover, it is unclear how one protein-RNA interaction might be transduced by RNA to kinetically impact a second. We report a parallelized, real-time single-molecule fluorescence assay for protein interaction kinetics on eukaryotic mRNA populations obtained from cells. We observed ∼100-fold heterogeneity for interactions of the translation initiation factor eIF4E with the universal mRNA 5' cap structure, dominated by steric effects on barrier-height variability for association. We also found that an RNA helicase, eIF4A, independently accelerated eIF4E-cap association. These data support a kinetic mechanism for how mRNA can determine the sensitivity of its translation to reduction in cellular eIF4E concentrations. They also support the view that global RNA structure significantly modulates protein-RNA interaction dynamics and can facilitate real-time communication between protein interactions at distinct sites.

Interaction between Transcription and Translation Machineries on a Nascent RNA with Higher Order Structure

During bacterial transcription, pausing of the RNA polymerase (RNAP) is an important feature that allows co-transcriptional RNA folding as well as protein recruitment. It has long been proposed that RNAP pausing in the vicinity of the translation start site allows the ribosome to bind the messenger RNA (mRNA) in order to couple the translation with the neo-synthesis of the transcript. There is a growing interest to understand the physical interaction between the ribosome and the RNAP in elongation complex (EC). Here, using both biochemical and single molecule fluorescence assays, we have investigated the mechanism of transcription-translation coupling of a pseudoknot-structured class 1 preQ1 riboswitch with higher order pseudoknot structure. Our results demonstrate that the RNAP significantly stabilizes the initial binding of the ribosome on the nascent riboswitch transcript. In addition, while using the preQ1 ligand that binds to the riboswitch with high affinity to form a folded conformation of the pseudoknot, we show that the conformational stability of the nascent RNA also plays important role for initiating the ribosome binding. Furthermore, our results indicate that transcriptional factor NusG, which is known to establish a bridge between the RNAP and the ribosome, stabilizes ribosome binding on the nascent RNA. Our transcription assays show that, interaction with the ribosome significantly stabilizes the RNAP pause at close to the translation start site. However, while translating, the ribosome releases the stalled RNAP showing that transcription-translation coupling is determinant for transcription progression. Currently we are working on a protein induced fluorescence enhancement (PIFE) assay to understand the effect of the rate of translation onto the rate of transcription by RNAP. These results provide significant insights to understand the cooperative mechanism of transcription and translation, and its effect on overall gene expression.

Pore Assembly of Bacterial Alpha Pore-Forming Toxin (αPFT), Cytolysin a on Lipid Membranes

Due to its significance as the primary barrier to cellular entry, cell membrane integrity is key to its sustenance. To overcome this barrier, pathogens have devised diverse cell membrane disruption strategies. Once such mechanism is the formation of nanopores by pore forming toxins (PFTs) released by several bacteria. PFTs, a major class of bacterial toxins are released as water soluble monomers. Upon binding the target bilayer membrane, they undergo structural rearrangements and oligomerize to form a stable transmembrane pore. The molecular mechanisms that result in efficient pore formation on eukaryotic membranes have been unclear. We dissect the assembly pathway of Cytolysin A (ClyA), a representative αPFT by devising single-molecule fluorescence assays that decouple pore formation stages and report on membrane binding, membrane insertion and oligomerization on artificial membrane bilayers. By evaluating the kinetics of intermediates along assembly pathway in membranes of different compositions, testing putative membrane binding domains with mutations, and combining them with mathematical modeling of erythrocyte lysis kinetics, we present a comprehensive mechanistic framework for ClyA pore formation. Our results show that cellular receptors are not essential for ClyA activity but cholesterol, a key component of mammalian membranes, is critical for cell lysis. Interestingly, initial binding of ClyA to membranes is not cholesterol dependent. However, cholesterol enhances the pore formation dynamics by driving the transmembrane domain insertion and oligomerization. Newly discovered cholesterol binding motifs within the N-terminus and β-tongue regions of the protein impart this selective enhancement of ClyA activity on cholesterol membranes. Finally, a cell lysis kinetics model which incorporates the cholesterol dependence of various pathway intermediates is developed to capture ClyA mediated erythrocyte lysis across different mutants.

Studying Tau-Microtubule Interaction Using Single-Molecule TIRF Microscopy.

Microtubule architecture depends on a complex network of microtubule-associated proteins (MAPs) that act in concert to modulate microtubule assembly/disassembly and spatial arrangement. In vitro reconstitution of cytoskeleton dynamics coupled to single-molecule fluorescence assays has opened new perspectives to quantify the interaction of MAPs with microtubules. Here, we present a Total Internal Reflection Fluorescence (TIRF) microscopy-based assay enabling the characterization of Tau interaction with dynamic microtubules at the single-molecule level. We describe protein sample preparation in flow cells, single-molecule acquisitions by TIRF microscopy, and quantitative analysis of Tau oligomerization states and dwell time on microtubules.