Eukaryotic DNA replication is initiated at multiple chromosomal sites known as origins of replication that are specifically recognized by the origin recognition complex (ORC) containing multiple ATPase sites. In budding yeast, ORC binds to specific DNA sequences known as autonomously replicating sequences (ARSs) that are mostly nucleosome depleted. However, nucleosomes may still inhibit the licensing of some origins by occluding ORC binding and subsequent MCM helicase loading. Using purified proteins and single-molecule visualization, we find here that the ORC can eject histones from a nucleosome in an ATP-dependent manner. The ORC selectively evicts H2A-H2B dimers but leaves the (H3-H4)2 tetramer on DNA. It also discriminates canonical H2A from the H2A.Z variant, evicting the former while retaining the latter. Finally, the bromo-adjacent homology (BAH) domain of the Orc1 subunit is essential for ORC-mediated histone eviction. These findings suggest that the ORC is a bona fide nucleosome remodeler that functions to create a local chromatin environment optimal for origin activity.

Rapid and accurate mRNA translation requires efficient codon-dependent delivery of the correct aminoacyl-tRNA (aa-tRNA) to the ribosomal A site. In mammals, this fidelity-determining reaction is facilitated by the GTPase elongation factor-1 alpha (eEF1A), which escorts aa-tRNA as an eEF1A(GTP)-aa-tRNA ternary complex into the ribosome. The structurally unrelated cyclic peptides didemnin B and ternatin-4 bind to the eEF1A(GTP)-aa-tRNA ternary complex and inhibit translation but have different effects on protein synthesis in vitro and in vivo. Here, we employ single-molecule fluorescence imaging and cryogenic electron microscopy to determine how these natural products inhibit translational elongation on mammalian ribosomes. By binding to a common site on eEF1A, didemnin B and ternatin-4 trap eEF1A in an intermediate state of aa-tRNA selection, preventing eEF1A release and aa-tRNA accommodation on the ribosome. We also show that didemnin B and ternatin-4 exhibit distinct effects on the dynamics of aa-tRNA selection that inform on observed disparities in their inhibition efficacies and physiological impacts. These integrated findings underscore the value of dynamics measurements in assessing the mechanism of small-molecule inhibition and highlight potential of single-molecule methods to reveal how distinct natural products differentially impact the human translation mechanism.

The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with re-analyses of genome-wide ORC binding data, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

Saccharomyces cerevisiae has been a faithful guide for study of eukaryotic DNA replication, as the numerous initiation and elongation proteins are conserved from yeast to human. However, there is a gap in our knowledge of why yeast uses a consensus DNA sequence at replication origins, while higher eukaryotes do not. The current study closes this gap. By direct single-molecule visualization, we show that the Origin Recognition Complex (ORC) searches for and stably binds nucleosomes, and that nucleosomes funtionalize ORC to load MCM helicase onto DNA, regardless of DNA sequence. Furthermore, we discover that ORC can remodel nucleosomes and expel H2A-H2B histone dimers, a heretofore unexpected function. Thus ORC helps create a chromatin environment permissive to origin function. The finding that ORC binding to nucleosomes leads to MCM loading at any DNA sequence is likely to generalize, and that higher eukaryotes follow this same paradigm for origin selection INTRODUCTION Complete and accurate duplication of the genome is critical for the proliferation of all organisms (1). The basic mechanism for the initiation of DNA replication is shared by all three domains of cellular life (2). An “initiator” first binds to a genomic site known as the origin of replication, then recruits the replicative helicase that is responsible for unwinding parental DNA duplex and creating templates for daughter strand synthesis. In eukaryotes, multiple origins across the genome are licensed for firing once, and only once, per cell cycle and their firing follows a temporally controlled program (3). The eukaryotic initiator is known as the origin recognition complex (ORC), which consists of six highly conserved subunits Orc1-6 (4, 5). ORC works in concert with Cdc6 and Cdt1 to coordinate the loading of two Mcm2-7 helicase complexes onto the origin (6, 7), forming the pre-replication complex (pre-RC) that potentially becomes activated during the subsequent S phase to produce bidirectional replication forks. In the model organism Saccharomyces cerevisiae (budding yeast), origins are located at a set of “replicator” positions known as autonomously replicating sequence (ARS) elements, each containing an AT-rich ARS consensus sequence (ACS) and other less conserved “B elements” (1). However, consensus ARS elements have yet to be found in higher eukaryotes including human (8). In fact, even in S. cerevisiae, the ARS and its internal ACS and B elements are not absolutely required for ORC binding, Mcm2-7 loading, or origin firing in vitro and in vivo (7, 9, 10). These observations indicate that there exist yet other chromosomal features that enable licensing and firing of eukaryotic replication origins (8, 11). One distinct challenge for ORC is the need to navigate through chromosomes predominantly packaged into nucleosomes (12), which influence multiple aspects of eukaryotic replication (13-15). Although still an area of active research, it is generally presumed that the nucleosome presents a barrier to ORC binding and origin activity, which must be overcome by DNA sequence or chromainremodeling enzymes. Consistent with the inhibitory effect of nucleosomes, it was shown that a nucleosome-free region (NFR) flanked by regularly positioned nucleosomes is a pronounced feature of ARS origin sites (16, 17). Notably, ORC binding and pre-RC assembly also play a direct role in shaping a local chromatin state permissive to replication initiation, often widening the NFR (18, 19). These studies suggest possible active participation of ORC in the formation of an adequate NFR for replication initiation. The interplay between ORC and nucleosomes is clearly a critical determinant for origin selection and function, but the molecular underpinning of this interplay remains incompletely understood. Single-molecule studies using reconstituted replication proteins have greatly aided our understanding of origin recognition and pre-RC formation (20-22). However, these studies have not examined the effect of nucleosomes on ORC activity. In this work, we employ correlative singlemolecule fluorescence and force microscopy, complemented with biochemical assays and genomic analysis, to interrogate the dynamic behavior of ORC on nucleosomal DNA. We find that, contrary to expectation, ORC searches for and actively remodels nucleosomes in a chromatinzed environment, and that nucleosomes are the primary determinant of origin recognition and licensing, regardless of the ARS DNA sequence. This finding of ARS-independent, nucleosome-dependent origin selection makes the yeast system much more like that expected for higher eukaryotes, and expands the role of ORC to one that can also help form the chromatin environment for initiation. RESULTS Direct observation of ORC interaction with nucleosomal DNA To perform the single-molecule studies we engineered a native S. cerevisiae origin sequence ARS1 (23) into the λ phage genome (Supplementary Fig. 1A), generating a DNA template (λARS1) that is biotinylated on both ends (Fig. 1A). We also purified the yeast Orc1-6 and Mcm2-7 complexes (referred to as ORC and MCM hereafter) (Supplementary Fig. 1B), as well as Cdc6 and Cdt1. To generate fluorescently labeled ORC for single-molecule visualization, we site-specifically attached a Cy3 fluorophore to the N terminus of the Orc1 subunit via a 12-residue peptide tag (S6), much smaller than the Halo-tag used in a recent single-molecule study (22). A single λARS1 molecule was tethered between a pair of streptavidin-coated beads in the microfluidic chamber of a dual-trap optical tweezers instrument also equipped with multicolor confocal fluorescence microscopy (24, 25) (Supplementary Fig. 1C). Upon moving the tethered DNA into a channel containing Cy3-ORC, Cdc6 and ATP, we observed ORC binding to DNA in real time (Fig. 1B). Consistent with previous results (21, 22), ORC-Cdc6 displayed diffusive behavior at non-ARS1 sites until it dissociated or encountered the ARS1 site, where the rapid diffusion behavior halted and, instead, ORC-Cdc6 resided stably (Fig. 1B). The distinctive behavior of ORC-Cdc6 at the engineered ARS1 site versus all other sites indicates that the λ genome does not contain a strong ACS motif, consistent with sequence analysis using a published algorithm (26) (Supplementary Fig. 2). Next, we set out to examine the behavior of ORC on nucleosomal DNA. We juxtaposed a Widom601 nucleosome positioning sequence next to the ARS1 site and generated the λ601ARS1 DNA template (Fig. 1C). We then loaded tethered λ601ARS1 with histone octamers in situ by the histone chaperone Nap1. This method has been previously used to form nucleosomes (14, 27) and also confirmed by us via single-molecule pulling experiments (Supplementary Fig. 3). The octamers were fluorescently labeled with Cy5 for direct visualization. We observed nucleosomes formed both at the 601 site next to ARS1 and at other locations (Fig. 1D), which allowed us to compare ORC-nucleosome interaction at ARS1 versus non-ARS1 sites. Surprisingly, we found that in the vast majority of cases ORC stably associated with the nucleosome, regardless of whether it was located adjacent to the ARS1 site or was at a non-ARS1 site (Fig. 1D). This is in contrast to the discrimination by ORC between ARS1 and non-ARS1 sites on non-nucleosomal DNA (Fig. 1E, F). Nucleosome targeting by ORC can conceivably be achieved by either a three-dimensional (3D) search (direct binding from solution) or a one-dimensional (1D) search (sliding along the DNA from a non-nucleosomal site). Indeed, we observed both modes in our data (Fig. 1D), with 3D search being the more dominant mode under our experimental conditions (Fig. 1G). These results suggest that ORC preferentially binds to nucleosomes when in a chromatinized

The Multidrug Resistance Protein 1 (MRP1) is an asymmetric ATP-binding cassette transporter that pumps diverse substrates out of the cell and confers resistance to chemotherapy. Structural studies have elucidated the inward- and outward-facing conformations of this transporter. However, the rate-limiting steps that govern the transport cycles of MRP1 remain unclear. We used single-molecule FRET to monitor the conformational dynamics of MRP1 during its active transport cycles. Combined with cryo-EM interrogation under active turnover conditions, we dissected the kinetic steps that control the transitions between inward- and outward-facing conformations.

ATP-binding cassette (ABC) transporters are molecular pumps ubiquitous across all kingdoms of life. While their structures have been widely reported, the kinetics governing their transport cycles remain largely unexplored. Multidrug resistance protein 1 (MRP1) is an ABC exporter that extrudes a variety of chemotherapeutic agents and native substrates. Previously, the structures of MRP1 were determined in an inward-facing (IF) or outward-facing (OF) conformation. Here, we used single-molecule fluorescence spectroscopy to track the conformational changes of bovine MRP1 (bMRP1) in real time. We also determined the structure of bMRP1 under active turnover conditions. Our results show that substrate stimulates ATP hydrolysis by accelerating the IF-to-OF transition. The rate-limiting step of the transport cycle is the dissociation of the nucleotide-binding-domain dimer, while ATP hydrolysis per se does not reset MRP1 to the resting state. The combination of structural and kinetic data illustrates how different conformations of MRP1 are temporally linked and how substrate and ATP alter protein dynamics to achieve active transport.

The eukaryotic replicative helicase CMG is a closed ring around double-stranded (ds)DNA at origins yet must transition to single-stranded (ss)DNA for helicase action. CMG must also handle repair intermediates, such as reversed forks that lack ssDNA. Here, using correlative single-molecule fluorescence and force microscopy, we show that CMG harbors a ssDNA gate that enables transitions between ss and dsDNA. When coupled to DNA polymerase, CMG remains on ssDNA, but when uncoupled, CMG employs this gate to traverse forked junctions onto dsDNA. Surprisingly, CMG undergoes rapid diffusion on dsDNA and can transition back onto ssDNA to nucleate a functional replisome. The gate—distinct from that between Mcm2/5 used for origin loading—is intrinsic to CMG; however, Mcm10 promotes strand passage by enhancing the affinity of CMG to DNA. This gating process may explain the dsDNA-to-ssDNA transition of CMG at origins and help preserve CMG on dsDNA during fork repair.

Adaptive immune systems must accurately distinguish between self and non-self in order to defend against invading pathogens while avoiding autoimmunity. Type III CRISPR-Cas systems employ guide RNA to recognize complementary RNA targets, which triggers the degradation of both the invader's transcripts and their template DNA. These systems can broadly eliminate foreign targets with multiple mutations but circumvent damage to the host genome. To explore the molecular basis for these features, we use single-molecule fluorescence microscopy to study the interaction between a type III-A ribonucleoprotein complex and various RNA substrates. We find that Cas10-the DNase effector of the complex-displays rapid conformational fluctuations on foreign RNA targets, but is locked in a static configuration on self RNA. Target mutations differentially modulate Cas10 dynamics and tune the CRISPR interference activity in vivo. These findings highlight the central role of the internal dynamics of CRISPR-Cas complexes in self versus non-self discrimination and target specificity.

ABSTRACTThe eukaryotic replicative helicase CMG is assembled at replication origins and is thought to remain topologically closed until termination. Upon encountering a lesion, CMG must vacate a stalled fork to allow DNA repair. However, the fate of CMG under these stress conditions remains unclear. Here, using correlative single-molecule fluorescence and force microscopy, we show that when uncoupled from a DNA polymerase, CMG opens a single-stranded (ss) DNA gate to traverse a forked junction and reside on double-stranded (ds) DNA. Surprisingly, CMG undergoes rapid diffusion on dsDNA and can transition back onto ssDNA for continued fork progression. The accessory protein Mcm10 is required for robust ssDNA gating. These results reveal an Mcm10-induced pathway that preserves CMG on DNA and allows it to access a repaired fork for swift replication recovery.

Prevailing dogma holds that ribosomes are uniform in composition and function. Single-molecule investigations of translation reveal, however, that the ribosome and translation factors visit metastable, transient intermediates that can be highly sensitive to even modest perturbations, including small-molecule drugs and single-nucleotide substitutions in ribosomal RNA (rRNA). These insights suggest the possibility that subtle changes in the ribosome's composition may have functional impacts. Recent evidence further implicates changes in the ribosome's core protein composition with gene-specific changes in translational efficiency. In this light, we set out to examine the potential physiological impacts of endogenously encoded sequence variations in the rRNA components of the assembled ribosome. Mammalian genomes encode hundreds of ribosomal DNA operons (rDNA) that exhibit extensive sequence variation with the rRNA components of the ribosome, which are both conserved and expressed in a tissue-specific fashion. Using E.coli as a genetically tractable model system, we now show that nutrient limitation-induced stress changes the relative expression of rDNA operons to alter the ribosomal RNA (rRNA) composition within the actively translating ribosome pool. The most upregulated operon encodes the unique 16S rRNA gene, rrsH, distinguished by conserved sequence variation within the small ribosomal subunit. rrsH-bearing ribosomes alter the levels of the RpoS sigma factor, the master regulator of the general stress response, to affect the expression of functionally coherent gene sets. These impacts are associated with phenotypic changes in antibiotic sensitivity, biofilm formation, and cell motility, and are regulated by ribosome-associated stress response proteins. Specific aspects of these phenotypic differences could be reconstituted in vitro using highly purified translation components. These findings establish that endogenously encoded, naturally occurring rRNA sequence variation can modulate ribosome function, central aspects of gene expression regulation, and cellular physiology.

DNA is both a fundamental building block of life and a fascinating natural polymer. The advent of single-molecule manipulation tools made it possible to exert controlled force on individual DNA molecules and measure their mechanical response. Such investigations elucidated the elastic properties of DNA and revealed its distinctive structural configurations across force regimes. In the meantime, a detailed understanding of DNA mechanics laid the groundwork for single-molecule studies of DNA-binding proteins and DNA-processing enzymes that bend, stretch, and twist DNA. These studies shed new light on the metabolism and transactions of nucleic acids, which constitute a major part of the cell's operating system. Furthermore, the marriage of single-molecule fluorescence visualization and force manipulation has enabled researchers to directly correlate the applied tension to changes in the DNA structure and the behavior of DNA-templated complexes. Overall, experimental exploitation of DNA mechanics has been and will continue to be a unique and powerful strategy for understanding how molecular machineries recognize and modify the physical state of DNA to accomplish their biological functions.

Single-molecule fluorescence microscopy is uniquely suited for detecting transient molecular recognition events, yet achieving the time resolution and statistics needed to realize this potential has proven challenging. Here we present a single-molecule imaging and analysis platform using scientific complementary metal-oxide semiconductor (sCMOS) detectors that enables imaging of 15,000 individual molecules simultaneously at millisecond rates. This system enabled the detection of previously obscured processes relevant to the fidelity mechanism in protein synthesis.

Directional translocation of the ribosome through the mRNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of tRNA and mRNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations revealed direct evidence of structurally and kinetically distinct late intermediates during substrate movement, whose resolution determines the rate of translocation. These steps involve intramolecular events within the EF-G-GDP-bound ribosome, including exaggerated, reversible fluctuations of the small-subunit head domain, which ultimately facilitate peptidyl-tRNA's movement into its final post-translocation position.

Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation have remained poorly understood. Moreover, the functions of modifications to ribosomal RNA and ribosomal proteins have also been unclear. Here we present the structure of the Escherichia coli 70S ribosome at 2.4-Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and it suggests how solvation contributes to ribosome integrity and function as well as how the conformation of ribosomal protein uS12 aids in mRNA decoding. This structure helps to explain the phylogenetic conservation of key elements of the ribosome, including post-transcriptional and post-translational modifications, and should serve as a basis for future antibiotic development.

Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin—paromomycin, ribostamycin and neamine—each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6′-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6′-substituent and the drug’s net positive charge. By solving the crystal structure of the paromomycin–ribosome complex, we observe specific contacts between the apical tip of H69 and the 6′-hydroxyl on paromomycin from within the drug’s canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation.

Background: Elongation factor-Tu (EF-Tu) chaperones aminoacyl-tRNA (aa-tRNA) to the elongating ribosome as a ternary complex with GTP. Results: EF-Ts facilitates EF-Tu·GTP binding to aa-tRNA through direct, transient interactions. Conclusion: EF-Ts accelerates the formation and decay of ternary complex through the formation of a transient EF-Tu/Ts·GTP·aa-tRNA quaternary complex. Significance: These newly described interactions of EF-Ts may serve to regulate ternary complex abundance in the cell. During protein synthesis, elongation factor-Tu (EF-Tu) bound to GTP chaperones the entry of aminoacyl-tRNA (aa-tRNA) into actively translating ribosomes. In so doing, EF-Tu increases the rate and fidelity of the translation mechanism. Recent evidence suggests that EF-Ts, the guanosine nucleotide exchange factor for EF-Tu, directly accelerates both the formation and dissociation of the EF-Tu-GTP-Phe-tRNAPhe ternary complex (Burnett, B. J., Altman, R. B., Ferrao, R., Alejo, J. L., Kaur, N., Kanji, J., and Blanchard, S. C. (2013) J. Biol. Chem. 288, 13917–13928). A central feature of this model is the existence of a quaternary complex of EF-Tu/Ts·GTP·aa-tRNAaa. Here, through comparative investigations of phenylalanyl, methionyl, and arginyl ternary complexes, and the development of a strategy to monitor their formation and decay using fluorescence resonance energy transfer, we reveal the generality of this newly described EF-Ts function and the first direct evidence of the transient quaternary complex species. These findings suggest that EF-Ts may regulate ternary complex abundance in the cell through mechanisms that are distinct from its guanosine nucleotide exchange factor functions.

Fluorescence provides a mechanism for achieving contrast in biological imaging that enables investigations of molecular structure, dynamics, and function at high spatial and temporal resolution. Small-molecule organic fluorophores have proven essential for such efforts and are widely used in advanced applications such as single-molecule and super-resolution microscopy. Yet, organic fluorophores, like all fluorescent species, exhibit instabilities in their emission characteristics, including blinking and photobleaching that limit their utility and performance. Here, we review the photophysics and photochemistry of organic fluorophores as they pertain to mitigating such instabilities, with a specific focus on the development of stabilized fluorophores through derivatization. Self-healing organic fluorophores, wherein the triplet state is intramolecularly quenched by a covalently attached protective agent, exhibit markedly improved photostabilities. We discuss the potential for further enhancements towards the goal of developing “ultra-stable” fluorophores spanning the visible spectrum and how such fluorophores are likely to impact the future of single-molecule research.

Single-molecule Förster resonance energy transfer (smFRET) is an essential and maturing tool to probe biomolecular interactions and conformational dynamics in vitro and, increasingly, in living cells. Multi-color smFRET enables the correlation of multiple such events and the precise dissection of their order and timing. However, the requirements for good spectral separation, high time resolution, and extended observation times place extraordinary demands on the fluorescent labels used in such experiments. Together with advanced experimental designs and data analysis, the development of long-lasting, non-fluctuating fluorophores is therefore proving key to progress in the field. Recently developed strategies for obtaining ultra-stable organic fluorophores spanning the visible spectrum are underway that will enable multi-color smFRET studies to deliver on their promise of previously unachievable biological insights.