231 research outputs found
Characterizing mRNA Interactions with RNA Granules during Translation Initiation Inhibition
When cells experience environmental stresses, global translational arrest is
often accompanied by the formation of stress granules (SG) and an increase in
the number of p-bodies (PBs), which are thought to play a crucial role in the
regulation of eukaryotic gene expression through the control of mRNA translation
and degradation. SGs and PBs have been extensively studied from the perspective
of their protein content and dynamics but, to date, there have not been
systematic studies on how they interact with native mRNA granules. Here, we
demonstrate the use of live-cell hybridization assays with multiply-labeled
tetravalent RNA imaging probes (MTRIPs) combined with immunofluorescence, as a
tool to characterize the polyA+ and β-actin mRNA distributions within
the cytoplasm of epithelial cell lines, and the changes in their colocalization
with native RNA granules including SGs, PBs and the RNA exosome during the
inhibition of translational initiation. Translation initiation inhibition was
achieved via the induction of oxidative stress using sodium arsenite, as well as
through the use of Pateamine A, puromycin and cycloheximide. This methodology
represents a valuable tool for future studies of mRNA trafficking and regulation
within living cells
Elementary simulation of tethered Brownian motion
We describe a simple numerical simulation, suitable for an undergraduate
project (or graduate problem set), of the Brownian motion of a particle in a
Hooke-law potential well. Understanding this physical situation is a practical
necessity in many experimental contexts, for instance in single molecule
biophysics; and its simulation helps the student to appreciate the dynamical
character of thermal equilibrium. We show that the simulation succeeds in
capturing behavior seen in experimental data on tethered particle motion.Comment: Submitted to American Journal of Physic
DNA Looping Kinetics Analyzed Using Diffusive Hidden Markov Model
Tethered particle experiments use light microscopy to measure the position of
a micrometer-sized bead tethered to a microscope slide via a ~micrometer length
polymer, in order to infer the behavior of the invisible polymer. Currently,
this method is used to measure rate constants of DNA loop formation and
breakdown mediated by repressor protein that binds to the DNA. We report a new
technique for measuring these rates using a modified hidden Markov analysis
that directly incorporates the diffusive motion of the bead, which is an
inherent complication of tethered particle motion because it occurs on a time
scale between the sampling frequency and the looping time. We compare looping
lifetimes found with our method, which are consistent over a range of sampling
frequencies, to those obtained via the traditional threshold-crossing analysis,
which vary depending on how the raw data are filtered in the time domain. Our
method does not involve such filtering, and so can detect short-lived looping
events and sudden changes in looping behavior.Comment: 3 page pdf including 3 figures corrections: 2nd page, 1st column,
values of diffusion coefficient, spring constant and the decay time were
typed incorrectly. No conlcusions were affecte
Tethered Particle Motion as a Diagnostic of DNA Tether Length
The tethered particle motion (TPM) technique involves an analysis of the Brownian motion of a bead tethered to a slide by a single DNA molecule. We describe an improved experimental protocol with which to form the tethers, an algorithm for analyzing bead motion visualized using differential interference contrast microscopy, and a physical model with which we have successfully simulated such DNA tethers. Both experiment and theory show that the statistics of the bead motion are quite different from those of a free semiflexible polymer. Our experimental data for chain extension versus tether length fit our model over a range of tether lengths from 109 to 3477 base pairs, using a value for the DNA persistence length that is consistent with those obtained under similar solution conditions by other methods. Moreover, we present the first experimental determination of the full probability distribution function of bead displacements and find excellent agreement with our theoretical prediction. Our results show that TPM is a useful tool for monitoring large conformational changes such as DNA looping
Calibration of Tethered Particle Motion Experiments
The Tethered Particle Motion (TPM) method has been used to observe and characterize a variety of protein-DNA interactions including DNA loping and transcription. TPM experiments exploit the Brownian motion of a DNA-tethered bead to probe biologically relevant conformational changes of the tether. In these experiments, a change in the extent of the bead’s random motion is used as a reporter of the underlying macromolecular dynamics and is often deemed sufficient for TPM analysis. However, a complete understanding of how the motion depends on the physical properties of the tethered particle complex would permit more quantitative and accurate evaluation of TPM data. For instance, such understanding can help extract details about a looped complex geometry (or multiple coexisting geometries) from TPM data. To better characterize the measurement capabilities of TPM experiments involving DNA tethers, we have carried out a detailed calibration of TPM magnitude as a function of DNA length and particle size. We also explore how experimental parameters such as acquisition time and exposure time affect the apparent motion of the tethered particle. We vary the DNA length from 200 bp to 2.6 kbp and consider particle diameters of 200, 490 and 970 nm. We also present a systematic comparison between measured particle excursions and theoretical expectations, which helps clarify both the experiments and models of DNA conformation
Diffusive hidden Markov model characterization of DNA looping dynamics in tethered particle experiments
In many biochemical processes, proteins bound to DNA at distant sites are
brought into close proximity by loops in the underlying DNA. For example, the
function of some gene-regulatory proteins depends on such DNA looping
interactions. We present a new technique for characterizing the kinetics of
loop formation in vitro, as observed using the tethered particle method, and
apply it to experimental data on looping induced by lambda repressor. Our
method uses a modified (diffusive) hidden Markov analysis that directly
incorporates the Brownian motion of the observed tethered bead. We compare
looping lifetimes found with our method (which we find are consistent over a
range of sampling frequencies) to those obtained via the traditional
threshold-crossing analysis (which can vary depending on how the raw data are
filtered in the time domain). Our method does not involve any time filtering
and can detect sudden changes in looping behavior. For example, we show how our
method can identify transitions between long-lived, kinetically distinct states
that would otherwise be difficult to discern
Spatial and topological organization of DNA chains induced by gene co-localization
Transcriptional activity has been shown to relate to the organization of
chromosomes in the eukaryotic nucleus and in the bacterial nucleoid. In
particular, highly transcribed genes, RNA polymerases and transcription factors
gather into discrete spatial foci called transcription factories. However, the
mechanisms underlying the formation of these foci and the resulting topological
order of the chromosome remain to be elucidated. Here we consider a
thermodynamic framework based on a worm-like chain model of chromosomes where
sparse designated sites along the DNA are able to interact whenever they are
spatially close-by. This is motivated by recurrent evidence that there exists
physical interactions between genes that operate together. Three important
results come out of this simple framework. First, the resulting formation of
transcription foci can be viewed as a micro-phase separation of the interacting
sites from the rest of the DNA. In this respect, a thermodynamic analysis
suggests transcription factors to be appropriate candidates for mediating the
physical interactions between genes. Next, numerical simulations of the polymer
reveal a rich variety of phases that are associated with different topological
orderings, each providing a way to increase the local concentrations of the
interacting sites. Finally, the numerical results show that both
one-dimensional clustering and periodic location of the binding sites along the
DNA, which have been observed in several organisms, make the spatial
co-localization of multiple families of genes particularly efficient.Comment: Figures and Supplementary Material freely available on
http://dx.doi.org/10.1371/journal.pcbi.100067
Nonspecific Protein-DNA Binding Is Widespread in the Yeast Genome
Recent genome-wide measurements of binding preferences of ~200 transcription
regulators in the vicinity of transcription start sites in yeast, have provided
a unique insight into the cis- regulatory code of a eukaryotic genome (Venters
et al., Mol. Cell 41, 480 (2011)). Here, we show that nonspecific transcription
factor (TF)-DNA binding significantly influences binding preferences of the
majority of transcription regulators in promoter regions of the yeast genome.
We show that promoters of SAGA-dominated and TFIID-dominated genes can be
statistically distinguished based on the landscape of nonspecific protein-DNA
binding free energy. In particular, we predict that promoters of SAGA-dominated
genes possess wider regions of reduced free energy compared to promoters of
TFIID-dominated genes. We also show that specific and nonspecific TF-DNA
binding are functionally linked and cooperatively influence gene expression in
yeast. Our results suggest that nonspecific TF-DNA binding is intrinsically
encoded into the yeast genome, and it may play a more important role in
transcriptional regulation than previously thought
High-throughput single-molecule analysis of DNA–protein interactions by tethered particle motion
Tethered particle motion (TPM) monitors the variations in the effective length of a single DNA molecule by tracking the Brownian motion of a bead tethered to a support by the DNA molecule. Providing information about DNA conformations in real time, this technique enables a refined characterization of DNA–protein interactions. To increase the output of this powerful but time-consuming single-molecule assay, we have developed a biochip for the simultaneous acquisition of data from more than 500 single DNA molecules. The controlled positioning of individual DNA molecules is achieved by self-assembly on nanoscale arrays fabricated through a standard microcontact printing method. We demonstrate the capacity of our biochip to study biological processes by applying our method to explore the enzymatic activity of the T7 bacteriophage exonuclease. Our single molecule observations shed new light on its behaviour that had only been examined in bulk assays previously and, more specifically, on its processivity
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