Papers by Ling Chin Hwang
ABSTRACTVibrio cholerae chromosome 2 (Chr2) requires its own ParABS system for segregation. Witho... more ABSTRACTVibrio cholerae chromosome 2 (Chr2) requires its own ParABS system for segregation. Without it, V. cholerae becomes nonviable and loses pathogenicity. ParA2 of Chr2 is a Walker-type ATPase that is the main driver of Chr2 segregation. Most of our understanding of ParA function comes from studying plasmid partition systems. How ParA provides the motive force in segregation of chromosomes, which are much larger than plasmids, is less understood and different models have been proposed. Here we analyzed in vivo behavior and kinetic properties of ParA2 using cell imaging, biochemical and biophysical approaches. ParA2 formed an asymmetric gradient in the cell that localized dynamically in the cell cycle. We found that ParA2 dimers bind ATP and undergo a slow conformational change to an active DNA-binding state, similar to P1 ParA. The presence of DNA catalyzes ParA2 conformational change to allow cooperative binding of active ParA2 dimers to form higher-order oligomers on DNA. Nucl...
Nature Communications, 2021
The efficient segregation of replicated genetic material is an essential step for cell division. ... more The efficient segregation of replicated genetic material is an essential step for cell division. Bacterial cells use several evolutionarily-distinct genome segregation systems, the most common of which is the type I Par system. It consists of an adapter protein, ParB, that binds to the DNA cargo via interaction with theparSDNA sequence; and an ATPase, ParA, that binds nonspecific DNA and mediates cargo transport. However, the molecular details of how this system functions are not well understood. Here, we report the cryo-EM structure of theVibrio choleraeParA2 filament bound to DNA, as well as the crystal structures of this protein in various nucleotide states. These structures show that ParA forms a left-handed filament on DNA, stabilized by nucleotide binding, and that ParA undergoes profound structural rearrangements upon DNA binding and filament assembly. Collectively, our data suggest the structural basis for ParA’s cooperative binding to DNA and the formation of high ParA dens...
Plasmid, 2017
Bacterial plasmid and chromosome segregation systems ensure that genetic material is efficiently ... more Bacterial plasmid and chromosome segregation systems ensure that genetic material is efficiently transmitted to progeny cells. Cell-based studies have shed light on the dynamic nature and the molecular basis of plasmid partition systems. In vitro reconstitutions, on the other hand, have proved to be an invaluable tool for studying the minimal components required to elucidate the mechanism of DNA segregation. This allows us to gain insight into the biological and biophysical processes that enable bacterial cells to move and position DNA. Here, we review the reconstitutions of plasmid partition systems in cell-free reactions, and discuss recent work that has begun to challenge long standing models of DNA segregation in bacteria.
Molecular cell, Sep 15, 2016
In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that... more 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 transitio...
Proceedings of the National Academy of Sciences of the United States of America, Jan 16, 2016
The Escherichia coli Min system self-organizes into a cell-pole to cell-pole oscillator on the me... more 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 releasi...
Biophysical Journal, 2014
Methods in Molecular Biology™, 2009
We present a single-molecule method for studying protein-DNA interactions based on fluorescence r... more 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.
Handbook of Single-Molecule Biophysics, 2009
Journal of Molecular Biology, 2013
Biophysical Journal, 2012
Biophysical Journal, 2011
Biophysical Journal, 2010
Biochemical Society Transactions, 2008
Single-molecule fluorescence methods, particularly single-molecule FRET (fluorescence resonance e... more 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.
Applied and Environmental Microbiology
With increasing environmental concerns about the exhausting use of fossil fuels, development of a... more With increasing environmental concerns about the exhausting use of fossil fuels, development of a sustainable biofuel production platform has been attracting significant public attention. Ethanologenic Z. mobilis species are endowed with an efficient ethanol fermentation capacity that surpasses, in several respects, that of baker’s yeast ( Saccharomyces cerevisiae ), the most-used microorganism for ethanol production.
Alphaproteobacterium Zymomonas mobilis exhibits extreme ethanologenic physiology, making this spe... more Alphaproteobacterium Zymomonas mobilis exhibits extreme ethanologenic physiology, making this species a promising biofuel producer. Numerous studies have investigated its biology relevant to industrial applications and mostly at the population level. However, the organization of single cells in this industrially important, polyploid species has been largely uncharacterized.In the present study, we characterized basic cellular behaviour of Z. mobilis strain Zm6 at a single cell level. We observed that growing Z. mobilis cells often divided at non mid-cell position, which contributed to variant cell size at birth. Yet, the cell size variance was regulated by a modulation of cell cycle span, mediated by a correlation of bacterial tubulin homologue FtsZ-ring accumulation with cell growth. The Z. mobilis culture also exhibited heterogeneous cellular DNA contents among individual cells, which might have been caused by asynchronous replication of chromosome that was not coordinated to cell...
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Papers by Ling Chin Hwang