Ling Chin Hwang

ParA-mediated plasmid partition driven by protein pattern self-organization

DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low‐copy plasmids, such as the plasmids P1 and F, employ a three‐component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker‐type ATPase, typically called ParA, which also binds non‐specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP‐driven patterning is involved in partition is unknown. We reconstituted and visualized ParA‐mediated plasmid partition inside a DNA‐carpeted flowcell, which acts as an artificial nucleoid. ParA and ParB transiently bridged plasmid to the DNA carpet. ParB‐stimulated ATP hydrolysis by ParA resulted in ParA disassembly from the bridging complex and from the surrounding DNA carpet, which led to plasmid detachment. Our results support a diffusion‐ratchet model, where ParB on the plasmid chases and redistributes the ParA gradient on the nucleoid, which in turn mobilizes the plasmid.

Cell-free study of F plasmid partition provides evidence for cargo transport by a diffusion-ratchet mechanism.

Significance ParA-type partition systems self-organize and pattern the bacterial nucleoid to organize plasmids, chromosomes, and protein machinery spatially. To study how protein patterns generate cargo movement, we reconstituted and visualized the partition system of F plasmid using a DNA-carpeted flowcell as an artificial nucleoid surface. We found that the partition proteins could bridge plasmid to the DNA carpet dynamically and mediate plasmid motion. Our data favor a diffusion-ratchet mechanism inherently different from classical motor protein or actin/microtubule filament-based transport. We expect surface-mediated patterning to become increasingly recognized as a means of intracellular transport in all kingdoms of life. Increasingly diverse types of cargo are being found to be segregated and positioned by ParA-type ATPases. Several minimalistic systems described in bacteria are self-organizing and are known to affect the transport of plasmids, protein machineries, and chromosomal loci. One well-studied model is the F plasmid partition system, SopABC. In vivo, SopA ATPase forms dynamic patterns on the nucleoid in the presence of the ATPase stimulator, SopB, which binds to the sopC site on the plasmid, demarcating it as the cargo. To understand the relationship between nucleoid patterning and plasmid transport, we established a cell-free system to study plasmid partition reactions in a DNA-carpeted flowcell. We observed depletion zones of the partition ATPase on the DNA carpet surrounding partition complexes. The findings favor a diffusion-ratchet model for plasmid motion whereby partition complexes create an ATPase concentration gradient and then climb up this gradient toward higher concentrations of the ATPase. Here, we report on the dynamic properties of the Sop system on a DNA-carpet substrate, which further support the proposed diffusion-ratchet mechanism.

Recent Advances in Fluorescence Cross-correlation Spectroscopy

Fluorescence cross-correlation spectroscopy (FCCS) is a method that measures the temporal fluorescence fluctuations coming from two differently labeled molecules diffusing through a small sample volume. Cross-correlation analysis of the fluorescence signals from separate detection channels extracts information of the dynamics of the dual-labeled molecules. FCCS has become an essential tool for the characterization of diffusion coefficients, binding constants, kinetic rates of binding, and determining molecular interactions in solutions and cells. By cross-correlating between two focal spots, flow properties could also be measured. Recent developments in FCCS have been targeted at using different experimental schemes to improve on the sensitivity and address their limitations such as cross-talk and alignment issues. This review presents an overview of the different excitation and detection methodologies used in FCCS and their biological applications. This is followed by a description of the fluorescent probes currently available for the different methods. This will introduce biological readers to FCCS and its related techniques and provide a starting point to selecting which experimental scheme is suitable for their type of biological study.

Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD

Significance The Min system of Escherichia coli uses the proteins MinD and MinE to form a standing wave oscillator on the membrane that prevents cell division at the cell poles. Using purified MinD and MinE, several dynamic patterns have been previously reconstituted on lipid bilayers. However, these dissimilar patterns occur under different reaction settings; therefore, the underlying mechanistic principles are unclear. By using a limiting supply of MinD, we reproduced standing wave oscillation on a flat bilayer. We find that periodic depletion of active MinD from solution is essential for the standing wave. Also, the MinD-to-MinE ratio on the bilayer acts as a toggle switch between membrane-binding and -release by MinD, which drives the oscillation. The Escherichia coli Min system self-organizes into a cell-pole to cell-pole oscillator on the membrane to prevent divisions at the cell poles. Reconstituting the Min system on a lipid bilayer has contributed to elucidating the oscillatory mechanism. However, previous in vitro patterns were attained with protein densities on the bilayer far in excess of those in vivo and failed to recapitulate the standing wave oscillations observed in vivo. Here we studied Min protein patterning at limiting MinD concentrations reflecting the in vivo conditions. We identified “burst” patterns—radially expanding and imploding binding zones of MinD, accompanied by a peripheral ring of MinE. Bursts share several features with the in vivo dynamics of the Min system including standing wave oscillations. Our data support a patterning mechanism whereby the MinD-to-MinE ratio on the membrane acts as a toggle switch: recruiting and stabilizing MinD on the membrane when the ratio is high and releasing MinD from the membrane when the ratio is low. Coupling this toggle switch behavior with MinD depletion from the cytoplasm drives a self-organized standing wave oscillator.

Single‐Molecule DNA Biosensors for Protein and Ligand Detection

Transcription factors (TFs) are sequence-specific DNA-binding proteins that control much of gene expression. TFs are natural biosensors and switches, translating chemical and physical signals (temperature shifts, light exposure, chemical concentrations, redox status) into transcriptional changes by modulating the binding of RNA polymerase to promoter DNA. Since changes in TF levels underlie fundamental biological processes such as DNA repair and cell-cycle progression, alterations in the levels of active TFs both lead to and indicate disease; for example, mutations in transcription factor p53 contribute to the rapid growth of cancer cells and, owing to their prevalence (p53 is mutated in roughly 50% of all human tumors), they have served as cancer biomarkers. Thus, methods for the sensitive detection and quantitation of TFs provide both fundamental information about gene regulation and a platform for diagnostics. TF detection often involves gel-based assays and Western blotting; although helpful in characterizing TF–DNA interactions, these assays are tedious, expensive, and qualitative, and consume large quantities of sample. Enzyme-linked immunosorbent assays (ELISAs) are more sensitive and offer higher throughput, but they require many preparation and signal-amplification steps for the detection of lowabundance TFs. Amplification is also required in the proximity-based ligation assay, making it incompatible with TF detection in living cells and diagnostic settings that demand results within minutes. An additional TF detection assay is based on fluorescence resonance energy transfer (FRET) between two doublestranded DNA (dsDNA) fragments containing fluorescently labeled single-stranded complementary overhangs (“molecular beacons”). In the presence of TF, the DNAs associate, resulting in donor fluorophore quenching as a result of FRET. This assay still requires significant amounts of sample and cannot detect low-abundance TFs; and because of the short dynamic range of FRET (1–10 nm), it also requires close proximity among the fluorophore, the quencher, and the protein–DNA interface, increasing the likelihood of steric interference with protein–DNA binding and complicating sensor design. Moreover, placing the fluorophore and the quencher on either side of the protein-binding site (usually 15–30 base pairs (bp) in length) on DNA results in very low FRET signals for most TFs. Here, we use alternating-laser excitation (ALEX) spectroscopy to detect TFs and small molecules by means of the TF-dependent coincidence of fluorescently labeled DNA. Like the molecular-beacon assay, our method is based on TFdriven DNA association, is rapid, and requires no amplification. However, our assay can detect pm levels of TFs in small amounts of sample, and it is FRET-independent, bypassing the need to optimize fluorophore position or know the structural details of TF–DNA binding; this flexibility in labeling ensures unperturbed TF–DNA binding. Using ALEX, we demonstrate TF and small-molecule detection, assay multiplexing, and suitability for analysis of complex biological samples. In our assay (Figure 1a,b), the full DNA-binding site for a TF is split in two (as in Ref. [5]): the left half-site (H1) and the right half-site (H2). Each site contains half of the TF-binding determinants and short, complementary 3’-overhangs. H1 is labeled with a “green” fluorophore (“G”) to give half-site H1, whereas H2 is labeled with a spectrally distinct “red” fluorophore (“R”) to give H2. In the absence of TF and at DNA concentrations of roughly 10–100 pm, H1 and H2 diffuse independently and associate only transiently. In contrast, in the presence of a TF that binds to the fully assembled DNA site, H1 and H2 diffuse as a complex (H1TF-H2; Figure 1a, bottom). We detect TF-dependent DNA coincidence using ALEX spectroscopy, wherein single molecules are excited by two lasers in an alternating fashion, with each laser capable of directly exciting either a G or a R fluorophore. ALEX allows molecular sorting on two-dimensional histograms of apparent FRET efficiency E* (a fluorescence ratio that reports on interfluorophore proximity) and probe stoichiometry S (a fluorescence ratio that reports on molecular stoichiometry). A search for all R-labeled molecules (i.e., G–R molecules [*] Dr. K. Lymperopoulos, R. Crawford, J. P. Torella, Dr. M. Heilemann, Dr. L. C. Hwang, S. J. Holden, Dr. A. N. Kapanidis Biological Physics Research Group, Department of Physics University of Oxford, Clarendon Laboratory Parks Road, Oxford, OX1 3PU (United Kingdom) E-mail: a.kapanidis1@physics.ox.ac.uk Dr. K. Lymperopoulos Current address: BioQuant Institute, Cellnetworks Cluster Ruprecht-Karls Universit t Heidelberg 69120 Heidelberg (Germany)

RNA Polymerase Pausing during Initial Transcription

Summary In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause (“initiation pause,” ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.

Red light, green light : probing single molecules using alternating-laser excitation

Single-molecule fluorescence methods, particularly single-molecule FRET (fluorescence resonance energy transfer), have provided novel insights into the structure, interactions and dynamics of biological systems. ALEX (alternating-laser excitation) spectroscopy is a new method that extends single-molecule FRET by providing simultaneous information about structure and stoichiometry; this new information allows the detection of interactions in the absence of FRET and extends the dynamic range of distance measurements that are accessible through FRET. In the present article, we discuss combinations of ALEX with confocal microscopy for studying in-solution and in-gel molecules; we also discuss combining ALEX with TIRF (total internal reflection fluorescence) for studying surface-immobilized molecules. We also highlight applications of ALEX to the study of protein-nucleic acid interactions.

Prism-based multicolor fluorescence correlation spectrometer

We report the design and application of a prism-based detection system for fluorescence (cross) correlation spectroscopy. The system utilizes a single laser wavelength for the simultaneous excitation of several dyes of different emission spectra. Fluorescence light is spectrally separated with a prismatic setup, and wavelengths are selected by scanning a fiber-coupled avalanche photodiode across the image spots. Multicolor autocorrelations are demonstrated with standard and tandem dyes, and fluorescence cross-correlation measurements of biotinylated nanocontainers and streptavidin are presented. This spectrometer offers high optical stability and no focal volume mismatch for the multicolor detection of molecular dynamics and interactions, with single-molecule sensitivity.

Single-Molecule FRET Analysis of Protein-DNA Complexes

We present a single-molecule method for studying protein-DNA interactions based on fluorescence resonance energy transfer (FRET) and alternating-laser excitation (ALEX) of single diffusing molecules. An application of this method to the study of a bacterial transcription initiation complex is presented.

Single-Molecule FRET: Methods and Biological Applications

Since the first single-molecule fluorescence resonance energy transfer (FRET) measurement in 1996, the technique has contributed substantially to our understanding of biological molecules and processes by probing the structure and dynamics of nucleic acids, protein molecules, and their complexes with other molecules. This review discusses basic concepts and current developments in single-molecule FRET methodology, as well as examples of applications to systems such as nucleic acid machines and molecular motors.