622 research outputs found
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
Concentration and Length Dependence of DNA Looping in Transcriptional Regulation
In many cases, transcriptional regulation involves the binding of transcription factors at sites on the DNA that are not immediately adjacent to the promoter of interest. This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter. These processes are important in settings ranging from the historic bacterial examples (bacterial metabolism and the lytic-lysogeny decision in bacteriophage), to the modern concept of gene regulation to regulatory processes central to pattern formation during development of multicellular organisms. Though there have been a variety of insights into the combinatorial aspects of transcriptional control, the mechanism of DNA looping as an agent of combinatorial control in both prokaryotes and eukaryotes remains unclear. We use single-molecule techniques to dissect DNA looping in the lac operon. In particular, we measure the propensity for DNA looping by the Lac repressor as a function of the concentration of repressor protein and as a function of the distance between repressor binding sites. As with earlier single-molecule studies, we find (at least) two distinct looped states and demonstrate that the presence of these two states depends both upon the concentration of repressor protein and the distance between the two repressor binding sites. We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA. The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the J-factor for looping
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
Changepoint Analysis for Single-Molecule Polarized Total Internal Reflection Fluorescence Microscopy Experiments
The experimental study of individual macromolecules has opened a door to determining the details of their mechanochemical operation. Motor enzymes such as the myosin family have been particularly attractive targets for such study, in part because some of them are highly processive and their “product” is spatial motion. But single-molecule resolution comes with its own costs and limitations. Often, the observations rest on single fluorescent dye molecules, which emit a limited number of photons before photobleaching and are subject to complex internal dynamics. Thus, it is important to develop methods that extract the maximum useful information from a finite set of detected photons. We have extended an experimental technique, multiple polarization illumination in total internal reflection fluorescence microscopy (polTIRF), to record the arrival time and polarization state of each individual detected photon. We also extended an analysis technique, previously applied to FRET experiments, that optimally determines times of changes in photon emission rates. Combining these improvements allows us to identify the structural dynamics of a molecular motor (myosin V) with unprecedented detail and temporal resolution
Twirling of Actin by Myosins II and V Observed via Polarized TIRF in a Modified Gliding Assay
The force generated between actin and myosin acts predominantly along the direction of the actin filament, resulting in relative sliding of the thick and thin filaments in muscle or transport of myosin cargos along actin tracks. Previous studies have also detected lateral forces or torques that are generated between actin and myosin, but the origin and biological role of these sideways forces is not known. Here we adapt an actin gliding filament assay in order to measure the rotation of an actin filament about its axis (“twirling”) as it is translocated by myosin. We quantify the rotation by determining the orientation of sparsely incorporated rhodamine-labeledactin monomers, using polarized total internal reflection (polTIRF) microscopy. In order to determine the handedness of the filament rotation, linear incident polarizations in between the standard s- and p-polarizations were generated, decreasing the ambiguity of our probe orientation measurement four-fold. We found that whole myosin II and myosin V both twirl actin with a relatively long (~ µm), left-handed pitch that is insensitive to myosin concentration, filament length and filament velocity
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
Twirling of actin by myosins II and V observed via polarized TIRF in a modified gliding assay
The force generated between actin and myosin acts predominantly along the
direction of the actin filament, resulting in relative sliding of the thick and
thin filaments in muscle or transport of myosin cargos along actin tracks.
Previous studies have also detected lateral forces or torques that are
generated between actin and myosin, but the origin and biological role of these
sideways forces is not known. Here we adapt an actin gliding filament assay in
order to measure the rotation of an actin filament about its axis (twirling) as
it is translocated by myosin. We quantify the rotation by determining the
orientation of sparsely incorporated rhodamine-labeled actin monomers, using
polarized total internal reflection (polTIRF) microscopy. In order to determine
the handedness of the filament rotation, linear incident polarizations in
between the standard s- and p-polarizations were generated, decreasing the
ambiguity of our probe orientation measurement four-fold. We found that whole
myosin II and myosin V both twirl actin with a relatively long (micron),
left-handed pitch that is insensitive to myosin concentration, filament length
and filament velocity
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
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
Isospin Purity of T=1 States in the A=38 Nuclei Studied Via Lifetime Measurements in K38
The Doppler Shift Attenuation Method was used to measure lifetimes for levels in 38K at excitation energies of 1698, 2404, 2830, 2996, and 3671 keV, populated using the 40Ca(d, α) 38K reaction at a beam energy of 4.5 MeV. Values of 109(29), 95(22), 457(63), 130(40), and 160(50) fs, respectively, were measured and are compared with previous values obtained using different stopping powers. The matrix element for the transition between the Jπ = 2+ T=1 and 0+ T=1 states in this Tz = 0 nucleus is compared with the analogous transition in the other nuclei in the T = 1 triplet, 38Ca (Tz = −1) and 38Ar (Tz = +1), and with the results of shell-model calculations
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