Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states - monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.

Precise control of actin filament length is essential to many cellular processes. Formins processively elongate filaments, whereas capping protein (CP) binds to barbed ends and arrests polymerization. While genetic and biochemical evidence has indicated that these two proteins function antagonistically, the mechanism underlying the antagonism has remained unresolved. Here we use multi-wavelength single-molecule fluorescence microscopy to observe the fully reversible formation of a long-lived 'decision complex' in which a CP dimer and a dimer of the formin mDia1 simultaneously bind the barbed end. Further, mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex. Quantitative kinetic analysis reveals that the CP-mDia1 antagonism that we observe in vitro occurs through the decision complex. Our observations suggest new molecular mechanisms for the control of actin filament length and for the capture of filament barbed ends in cells.

Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 are well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. Phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO-complex formation and provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO-complex formation.

Licensing of eukaryotic origins of replication requires DNA loading of two copies of the Mcm2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally. To understand the formation and importance of this double-hexamer, we identified mutations in a conserved and essential Mcm4 motif that permit loading of two Mcm2-7 complexes but are defective for double-hexamer formation. Single-molecule studies show mutant Mcm2-7 forms initial hexamer-hexamer interactions; however, the resulting complex is unstable. Kinetic analyses of wild-type and mutant Mcm2-7 reveal a limited time window for double-hexamer formation following second Mcm2-7 association, suggesting that this process is facilitated. Double-hexamer formation is required for extensive origin DNA unwinding but not initial DNA melting or recruitment of helicase-activation proteins (Cdc45, GINS, Mcm10). Our findings elucidate dynamic mechanisms of origin licensing, and identify the transition between initial DNA melting and extensive unwinding as the first initiation event requiring double-hexamer formation.

DNA transcription initiates after an RNA polymerase (RNAP) molecule binds to the promoter of a gene. In bacteria, the canonical picture is that RNAP comes from the cytoplasmic pool of freely diffusing RNAP molecules. Recent experiments suggest the possible existence of a separate pool of polymerases, competent for initiation, which freely slide on the DNA after having terminated one round of transcription. Promoter-dependent transcription reinitiation from this pool of post-termination RNAP may lead to coupled initiation at nearby operons, but it is unclear whether this can occur over the distance- and time-scales needed for it to function widely on a bacterial genome in vivo. Here, we mathematically model the hypothesized reinitiation mechanism as a diffusion-to-capture process and compute the distances over which significant inter-operon coupling can occur and the time required. These quantities depend on previously uncharacterized molecular association and dissociation rate constants between DNA, RNAP and the transcription initiation factor σ 70 ; we measure these rate constants using single-molecule experiments in vitro. Our combined theory/experimental results demonstrate that efficient coupling can occur at physiologically relevant σ 70 concentrations and on timescales appropriate for transcript synthesis. Coupling is efficient over terminator-promoter distances up to ∼ 1, 000 bp, which includes the majority of terminator-promoter nearest neighbor pairs in the E. coli genome. The results suggest a generalized mechanism that couples the transcription of nearby operons and breaks the paradigm that each binding of RNAP to DNA can produce at most one messenger RNA.

During origin licensing, the eukaryotic replicative helicase Mcm2-7 forms head-to-head double hexamers to prime origins for bidirectional replication. Recent single-molecule and structural studies revealed that one molecule of the helicase loader ORC can sequentially load two Mcm2-7 hexamers to ensure proper head-to-head helicase alignment. To perform this task, ORC must release from its initial high-affinity DNA binding site and "flip" to bind a weaker, inverted DNA site. However, the mechanism of this binding-site switch remains unclear. In this study, we used single-molecule Förster resonance energy transfer (sm-FRET) to study the changing interactions between DNA and ORC or Mcm2-7. We found that the loss of DNA bending that occurs during DNA deposition into the Mcm2-7 central channel increases the rate of ORC dissociation from DNA. Further studies revealed temporally-controlled DNA sliding of helicase-loading intermediates, and that the first sliding complex includes ORC, Mcm2-7, and Cdt1. We demonstrate that sequential events of DNA unbending, Cdc6 release, and sliding lead to a stepwise decrease in ORC stability on DNA, facilitating ORC dissociation from its strong binding site during site switching. In addition, the controlled sliding we observed provides insight into how ORC accesses secondary DNA binding sites at different locations relative to the initial binding site. Our study highlights the importance of dynamic protein-DNA interactions in the loading of two oppositely-oriented Mcm2-7 helicases to ensure bidirectional DNA replication.

The molecular basis for regulation of lactose metabolism in Escherichia coli is well studied. Nonetheless, the physical mechanism by which the Lac repressor protein prevents transcription of the lactose promoter remains unresolved. Using multi-wavelength single-molecule fluorescence microscopy, we visualized individual complexes of fluorescently tagged RNA polymerase holoenzyme bound to promoter DNA. Quantitative analysis of the single-molecule observations, including use of a novel statistical partitioning approach, reveals highly kinetically stable binding of polymerase to two different sites on the DNA, only one of which leads to transcription. Addition of Lac repressor directly demonstrates that bound repressor prevents the formation of transcriptionally productive open promoter complexes; discrepancies in earlier studies may be attributable to transcriptionally inactive polymerase binding. The single-molecule statistical partitioning approach is broadly applicable to elucidating mechanisms of regulatory systems including those that are kinetically rather than thermodynamically controlled.

Most human genes contain multiple introns, necessitating mechanisms to effectively define exons and ensure their proper connection by spliceosomes. Human spliceosome assembly involves both cross-intron and cross-exon interactions, but how these work together is unclear. We examined in human nuclear extracts dynamic interactions of single pre-mRNA molecules with individual fluorescently tagged spliceosomal subcomplexes to investigate how cross-intron and cross-exon processes jointly promote pre-spliceosome assembly. U1 subcomplex bound to the 5' splice site of an intron acts jointly with U1 bound to the 5' splice site of the next intron to dramatically increase the rate and efficiency by which U2 subcomplex is recruited to the branch site/3' splice site of the upstream intron. The flanking 5' splice sites have greater than additive effects implying distinct mechanisms facilitating U2 recruitment. This synergy of 5' splice sites across introns and exons is likely important in promoting correct and efficient splicing of multi-intron pre-mRNAs.

Cell locomotion and endocytosis are powered by the rapid polymerization and turnover of branched actin filament networks nucleated by Arp2/3 complex. Although a large number of cellular factors have been identified that stimulate Arp2/3 complex-mediated actin nucleation, only a small number of studies so far have addressed which factors promote actin network debranching. Here, we investigated the function of a conserved homolog of ADF/cofilin, glia maturation factor (GMF). We found that S. cerevisiae GMF (also called Aim7) localizes in vivo to cortical actin patches and displays synthetic genetic interactions with ADF/cofilin. However, GMF lacks detectable actin binding or severing activity and instead binds tightly to Arp2/3 complex. Using in vitro evanescent wave microscopy, we demonstrated that GMF potently stimulates debranching of actin filaments produced by Arp2/3 complex. Further, GMF inhibits nucleation of new daughter filaments. Together, these data suggest that GMF binds Arp2/3 complex to both "prune" daughter filaments at the branch points and inhibit new actin assembly. These activities and its genetic interaction with ADF/cofilin support a role for GMF in promoting the remodeling and/or disassembly of branched networks. Therefore, ADF/cofilin and GMF, members of the same superfamily, appear to have evolved to interact with actin and actin-related proteins, respectively, and to make mechanistically distinct contributions to the remodeling of cortical actin structures.

Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryote Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so is unclear. We used multi-wavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the E. coli transcription cycle. In our experiments, RapA at < 5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post-termination complex (PTC) -- consisting of core RNA polymerase (RNAP) bound to dsDNA -- and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription re-initiation in proteobacterial genomes.

Formation and turnover of branched actin networks underlies cell migration and other essential force-driven processes. Type I nucleation-promoting factors (NPFs) such as WASP recruit actin monomers to Arp2/3 complex to stimulate nucleation. In contrast, mechanisms of type II NPFs such as Abp1 (also known as HIP55 and Drebrin-like protein) are less well understood. Here, we use single-molecule analysis to investigate yeast Abp1 effects on Arp2/3 complex, and find that Abp1 strongly enhances Arp2/3-dependent branch nucleation by stabilizing Arp2/3 on sides of mother filaments. Abp1 binds dynamically to filament sides, with sub-second lifetimes, yet associates stably with branch junctions. Further, we uncover a role for Abp1 in protecting filament junctions from GMF-induced debranching by competing with GMF for Arp2/3 binding. These data, combined with EM structures of Abp1 dimers bound to Arp2/3 complex in two different conformations, expand our mechanistic understanding of type II NPFs.

The committed step of eukaryotic DNA replication occurs when the pairs of Mcm2-7 replicative helicases that license each replication origin are activated. Helicase activation requires the recruitment of Cdc45 and GINS to Mcm2-7, forming Cdc45-Mcm2-7-GINS complexes (CMGs). Using single-molecule biochemical assays to monitor CMG formation, we found that Cdc45 and GINS are recruited to loaded Mcm2-7 in two stages. Initially, Cdc45, GINS, and likely additional proteins are recruited to unstructured Mcm2-7 N-terminal tails in a Dbf4-dependent kinase (DDK)-dependent manner, forming Cdc45-tail-GINS intermediates (CtGs). DDK phosphorylation of multiple phosphorylation sites on the Mcm2-7 tails modulates the number of CtGs formed per Mcm2-7. In a second, inefficient event, a subset of CtGs transfer their Cdc45 and GINS components to form CMGs. Importantly, higher CtG multiplicity increases the frequency of CMG formation. Our findings reveal the molecular mechanisms sensitizing helicase activation to DDK levels with implications for control of replication origin efficiency and timing.

The opening and closing of two ring-shaped Mcm2-7 DNA helicases is necessary to license eukaryotic origins of replication, although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process by establishing a topological link between each Mcm2-7 hexamer and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule Förster resonance energy transfer (FRET), we monitored ring opening and closing of Saccharomyces cerevisiae Mcm2-7 during origin licensing. The two Mcm2-7 rings were open during initial DNA association and closed sequentially, concomitant with the release of their associated Cdt1. We observed that ATP hydrolysis by Mcm2-7 was coupled to ring closure and Cdt1 release, and failure to load the first Mcm2-7 prevented recruitment of the second Mcm2-7. Our findings identify key mechanisms controlling the Mcm2-7 DNA-entry gate during origin licensing, and reveal that the two Mcm2-7 complexes are loaded via a coordinated series of events with implications for bidirectional replication initiation and quality control.

The mechanisms by which cells destabilize and rapidly disassemble filamentous actin networks have remained elusive; however, Coronin, Cofilin and AIP1 have been implicated in this process. Here using multi-wavelength single-molecule fluorescence imaging, we show that mammalian Cor1B, Cof1 and AIP1 work in concert through a temporally ordered pathway to induce highly efficient severing and disassembly of actin filaments. Cor1B binds to filaments first, and dramatically accelerates the subsequent binding of Cof1, leading to heavily decorated, stabilized filaments. Cof1 in turn recruits AIP1, which rapidly triggers severing and remains bound to the newly generated barbed ends. New growth at barbed ends generated by severing was blocked specifically in the presence of all three proteins. This activity enabled us to reconstitute and directly visualize single actin filaments being rapidly polymerized by formins at their barbed ends while simultanteously being stochastically severed and capped along their lengths, and disassembled from their pointed ends.

Removal of introns from nascent transcripts (pre-mRNAs) by the spliceosome is an essential step in eukaryotic gene expression. Previous studies have suggested that the earliest steps in spliceosome assembly in yeast are highly ordered and the stable recruitment of U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site necessarily precedes recruitment of U2 snRNP to the branch site to form the "prespliceosome." Here, using colocalization single-molecule spectroscopy to follow initial spliceosome assembly on eight different S. cerevisiae pre-mRNAs, we demonstrate that active yeast spliceosomes can form by both U1-first and U2-first pathways. Both assembly pathways yield prespliceosomes functionally equivalent for subsequent U5·U4/U6 tri-snRNP recruitment and for intron excision. Although fractional flux through the two pathways varies on different introns, both are operational on all introns studied. Thus, multiple pathways exist for assembling functional spliceosomes. These observations provide insight into the mechanisms of cross-intron coordination of initial spliceosome assembly.

RNA polymerases (RNAPs) contain a conserved 'secondary channel' which binds regulatory factors that modulate transcription initiation. In Escherichia coli, the secondary channel factors (SCFs) GreB and DksA both repress ribosomal RNA (rRNA) transcription, but SCF loading and repression mechanisms are unclear. We observed in vitro fluorescently labeled GreB molecules binding to single RNAPs and initiation of individual transcripts from an rRNA promoter. GreB arrived and departed from promoters only in complex with RNAP. GreB did not alter initial RNAP-promoter binding but instead blocked a step after conformational rearrangement of the initial RNAP-promoter complex. Strikingly, GreB-RNAP complexes never initiated at an rRNA promoter; only RNAP molecules arriving at the promoter without bound GreB produced transcript. The data reveal that a model SCF functions by a 'delayed inhibition' mechanism and suggest that rRNA promoters are inhibited by GreB/DksA because their short-lived RNAP complexes do not allow sufficient time for SCFs to dissociate.

Cellular actin networks grow by ATP-actin addition at filament barbed ends and have long been presumed to depolymerize at their pointed ends, primarily after filaments undergo "aging" (ATP hydrolysis and Pi release). The cytosol contains high levels of actin monomers, which favors assembly over disassembly, and barbed ends are enriched in ADP-Pi actin. For these reasons, the potential for a barbed end depolymerization mechanism in cells has received little attention. Here, using microfluidics-assisted TIRF microscopy, we show that mouse twinfilin, a member of the ADF-homology family, induces depolymerization of ADP-Pi barbed ends even under assembly-promoting conditions. Indeed, we observe in single reactions containing micromolar concentrations of actin monomers the simultaneous rapid elongation of formin-bound barbed ends and twinfilin-induced depolymerization of free barbed ends. The data show that twinfilin catalyzes dissociation of subunits from ADP-Pi barbed ends and thereby bypasses filament aging prerequisites to disassemble newly polymerized actin filaments.

RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. Here we use single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remains bound to DNA and sometimes exhibits one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarts transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this secondary initiation in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation.

Eukaryotic cells contain branched actin networks that are essential for endocytosis, motility, and other key cellular processes. These networks, which are formed by filamentous actin and the Arp2/3 complex, must subsequently be debranched to allow network remodeling and to recycle the Arp2/3 complex. Debranching appears to be catalyzed by two different members of the actin depolymerizing factor homology protein family: cofilin and glial maturation factor (GMF). However, their mechanisms of debranching are only partially understood. Here, we used single-molecule fluorescence imaging of Arp2/3 complex and actin filaments under physiological ionic conditions to observe debranching by GMF and cofilin. We demonstrate that cofilin, like GMF, is an authentic debrancher independent of its filament-severing activity and that the debranching activities of the two proteins are additive. While GMF binds directly to the Arp2/3 complex, cofilin selectively accumulates on branch-junction daughter filaments in tropomyosin-decorated networks just prior to debranching events. Quantitative comparison of debranching rates with the known kinetics of cofilin-actin binding suggests that cofilin occupancy of a particular single actin site at the branch junction is sufficient to trigger debranching. In rare cases in which the order of departure could be resolved during GMF- or cofilin-induced debranching, the Arp2/3 complex left the branch junction bound to the pointed end of the daughter filament, suggesting that both GMF and cofilin can work by destabilizing the mother filament-Arp2/3 complex interface. Taken together, these observations suggest that GMF and cofilin promote debranching by distinct yet complementary mechanisms.

Replication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires origin-recognition complex (ORC), Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with single-molecule Förster resonance energy transfer (FRET), we investigated interactions between ORC and Mcm2-7 during helicase loading. In the large majority of events, we observed a single ORC molecule recruiting both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between first- and second-helicase recruitment, a rapid change in interactions between ORC and the first Mcm2-7 occurs. Within seconds, ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly coordinated series of events through which a single ORC molecule can load two oppositely oriented helicases.