tethered particle motion
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AbstractDuring elongation, RNA polymerase (RNAP) must navigate through proteins that decorate genomic DNA. Several of these mediate long-distance interactions via structures, such as loops, that alter DNA topology and create torsional barriers. We used the tethered particle motion (TPM) technique and magnetic tweezers to monitor transcription of DNA templates in the presence of the lac repressor (LacI) protein which could bind at two sites, one proximal to, and one distal from, the promoter. The bivalent LacI tetramer binds recognition sites (operators) with up to nanomolar affinity depending on the sequence, and the concentration of LacI was adjusted to promote binding to either one or both operators, so as to produce unlooped or looped DNA. We observed that RNAP pausing before a LacI-securing loop was determined not by the affinity of LacI for the operator, but by the order in which the elongating RNAP encountered these operators. TPM experiments showed that, independent of affinity, LacI bound at the promoter-proximal operator became a stronger roadblock when securing a loop. In contrast, LacI bound to the distal operator was a weaker roadblock in a looped configuration suggesting that RNAP might more easily displace LacI obstacles within a torsion-constrained DNA loop. Since protein junctions can efficiently block the diffusion of DNA supercoiling, these data indicate that the positive supercoiling generated ahead of a transcribing RNAP may facilitate the dissociation of a roadblock. In support of this idea, magnetic tweezers measurements indicated that pauses are shorter when RNAP encounters obstacles on positively supercoiled than on relaxed DNA. Furthermore, at similar winding levels of the DNA template, RNAP pause duration decreased with tension. These findings are significant for our understanding of transcription within the crowded and tensed nucleoid.

HU is a nucleoid-associated protein expressed in most eubacteria at a high amount of copies (tens of thousands). The protein is believed to bind across the genome to organize and compact the DNA. Most of the studies on HU have been carried out in a simple in vitro system, and to what extent these observations can be extrapolated to a living cell is unclear. In this study, we investigate the DNA binding properties of HU under conditions approximating physiological ones. We report that these properties are influenced by both macromolecular crowding and salt conditions. We use three different crowding agents (blotting grade blocker (BGB), bovine serum albumin (BSA), and polyethylene glycol 8000 (PEG8000)) as well as two different MgCl2 conditions to mimic the intracellular environment. Using tethered particle motion (TPM), we show that the transition between two binding regimes, compaction and extension of the HU protein, is strongly affected by crowding agents. Our observations suggest that magnesium ions enhance the compaction of HU–DNA and suppress filamentation, while BGB and BSA increase the local concentration of the HU protein by more than 4-fold. Moreover, BGB and BSA seem to suppress filament formation. On the other hand, PEG8000 is not a good crowding agent for concentrations above 9% (w/v), because it might interact with DNA, the protein, and/or surfaces. Together, these results reveal a complex interplay between the HU protein and the various crowding agents that should be taken into consideration when using crowding agents to mimic an in vivo system.

AbstractWe demonstrate an ultra-stable, highly dense single-molecule assay ideal for observing protein-DNA interactions. Stable click Tethered Particle Motion (scTPM) leverages next generation click-chemistry to achieve an ultrahigh density of surface tethered reporter particles, has a high antifouling resistance, is stable at elevated temperatures to at least 45 °C, and is compatible with Mg2+, an important ionic component of many regulatory protein-DNA interactions. Prepared samples remain stable, with little degradation, for > 6 months in physiological buffers. These improvements enabled us to study previously inaccessible sequence and temperature dependent effects on DNA binding by the bacterial protein H-NS, a global transcriptional regulator found in E. Coli. This greatly improved assay can directly be translated to accelerate existing tethered particle based, single-molecule biosensing applications.

Abstract Developing lymphocytes of jawed vertebrates cleave and combine distinct gene segments to assemble antigen–receptor genes. This process called V(D)J recombination that involves the RAG recombinase binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG–RSS interaction. We employ a single-molecule method known as tethered particle motion to track the formation, lifetime and cleavage of individual RAG–12RSS–23RSS paired complexes (PCs) for numerous synthetic and endogenous 12RSSs. We reveal that single-bp changes, including in the 12RSS spacer, can significantly and selectively alter PC formation or the probability of RAG-mediated cleavage in the PC. We find that some rarely used endogenous gene segments can be mapped directly to poor RAG binding on their adjacent 12RSSs. Finally, we find that while abrogating RSS nicking with Ca2+ leads to substantially shorter PC lifetimes, analysis of the complete lifetime distributions of any 12RSS even on this reduced system reveals that the process of exiting the PC involves unidentified molecular details whose involvement in RAG–RSS dynamics are crucial to quantitatively capture kinetics in V(D)J recombination.

Dmc1 recombinases are essential to homologous recombination in meiosis. Here, we studied the kinetics of the nucleoprotein filament assembly ofSaccharomyces cerevisiaeDmc1 using single-molecule tethered particle motion experiments and in vitro biochemical assay. ScDmc1 nucleoprotein filaments are less stable than the ScRad51 ones because of the kinetically much reduced nucleation step. The lower nucleation rate of ScDmc1 results from its lower single-stranded DNA (ssDNA) affinity, compared to that of ScRad51. Surprisingly, ScDmc1 nucleates mostly on the DNA structure containing the single-stranded and duplex DNA junction with the allowed extension in the 5′-to-3′ polarity, while ScRad51 nucleation depends strongly on ssDNA lengths. This nucleation preference is also conserved for mammalian RAD51 and DMC1. In addition, ScDmc1 nucleation can be stimulated by short ScRad51 patches, but not by EcRecA ones. Pull-down experiments also confirm the physical interactions of ScDmc1 with ScRad51 in solution, but not with EcRecA. Our results are consistent with a model that Dmc1 nucleation can be facilitated by a structural component (such as DNA junction and protein–protein interaction) and DNA polarity. They provide direct evidence of how Rad51 is required for meiotic recombination and highlight a regulation strategy in Dmc1 nucleoprotein filament assembly.

AbstractRegulated proteolysis of signaling proteins under mechanical tension enables cells to communicate with their environment in a variety of developmental and physiologic contexts. The role of force in inducing proteolytic sensitivity has been explored using magnetic tweezers at the single-molecule level with bead-tethered assays, but such efforts have been limited by challenges in ensuring that beads are not restrained by multiple tethers. Here, we describe a multiplexed assay for single-molecule proteolysis that overcomes the multiple-tether problem using a flow extension (FLEX) strategy on a microscope equipped with magnetic tweezers. Particle tracking and computational sorting of flow-induced displacements allows assignment of tethered substrates into singly-captured and multiply-tethered bins, with the fraction of fully mobile, single-tethered substrates depending inversely on the concentration of substrate loaded on the coverslip. Computational exclusion of multiply-tethered beads enables robust assessment of on-target proteolysis by the highly specific tobacco etch virus protease and the more promiscuous metalloprotease ADAM17. This method should be generally applicable to a wide range of proteases and readily extensible to robust evaluation of proteolytic sensitivity as a function of applied magnetic force.