Tethered Particle Motion Method Research Articles

Studying Protein-DNA Dynamics and Protein Unfolding Using a Force-Free Single-Molecule Technique

We are using optimized variations of a force-free single-molecule technique, known as Tethered Particle Motion (TPM) to study protein-DNA dynamics and protein folding/refolding in their natural form. First we use it to quantitatively describe how HU protein alters the DNA persistence length with regard to its concentration by locally bending or unbending different regions in the DNA and hence, changing the local flexibility of the DNA chain. A second variation of the TPM method enables us to track the folding and unfolding of single polypeptide chains composed of I27 tandem repeats using either chemical denaturation (force-free) or force unfolding using flow or AFM.

HU Protein Induces Incoherent DNA Persistence Length

HU is a highly conserved protein that is believed to play an important role in the architecture and dynamic compaction of bacterial DNA. Its ability to control DNA bending is crucial for functions such as transcription and replication. The effects of HU on the DNA structure have been studied so far mainly by single molecule methods that require us to apply stretching forces on the DNA and therefore may perturb the DNA-protein interaction. To overcome this hurdle, we study the effect of HU on the DNA structure without applying external forces by using an improved tethered particle motion method. By combining the results with DNA curvature analysis from atomic force microscopy measurements we find that the DNA consists of two different curvature distributions and the measured persistence length is determined by their interplay. As a result, the effective persistence length adopts a bimodal property that depends primarily on the HU concentration. The results can be explained according to a recently suggested model that distinguishes single protein binding from cooperative protein binding.

Force-free measurements of the conformations of DNA molecules tethered to a wall

Using an optimized combination of tethered particle motion method, total internal reflection, and a gold nanobead, we measured the three-dimensional distribution of the free end of a tethered DNA molecule. The distribution along the axial z direction (perpendicular to the surface) is found to be Rayleigh-like, in agreement with wormlike chain and freely jointed chain simulations. Using these simulations, we show that the presence of the wall increases the correlations between the orientations of neighboring chain segments compared to free DNA. While the measured and the simulated planar (xy) distributions always agree with that of a Gaussian-random-walk (GRW) model, for short DNA lengths (1 μm) studied in our experiment, the corresponding axial (z) distributions deviate from those predicted for a GRW confined to half-space.

Single-Molecule TPM Studies on the Conversion of Human Telomeric DNA

Human telomere contains guanine-rich (G-rich) tandem repeats of single-stranded DNA sequences at its 3′ tail. The G-rich sequences can be folded into various secondary structures, termed G-quadruplexes (G4s), by Hoogsteen basepairing in the presence of monovalent cations (such as Na+ and K+). We developed a single-molecule tethered particle motion (TPM) method to investigate the unfolding process of G4s in the human telomeric sequence AGGG(TTAGGG)3 in real time. The TPM method monitors the DNA tether length change caused by formation of the G4, thus allowing the unfolding process and structural conversion to be monitored at the single-molecule level. In the presence of its antisense sequence, the folded G4 structure can be disrupted and converted to the unfolded conformation, with apparent unfolding time constants of 82 s and 3152 s. We also observed that the stability of the G4 is greatly affected by different monovalent cations. The folding equilibrium constant of G4 is strongly dependent on the salt concentration, ranging from 1.75 at 5 mM Na+ to 3.40 at 15 mM Na+. Earlier spectral studies of Na+- and K+-folded states suggested that the spectral conversion between these two different folded structures may go through a structurally unfolded intermediate state. However, our single-molecule TPM experiments did not detect any totally unfolded intermediate within our experimental resolution when sodium-folded G4 DNA molecules were titrated with high-concentration, excess potassium ions. This observation suggests that a totally unfolding pathway is likely not the major pathway for spectral conversion on the timescale of minutes, and that interconversion among folded states can be achieved by the loop rearrangement. This study also demonstrates that TPM experiments can be used to study conformational changes in single-stranded DNA molecules.

Investigating the Nucleation and Extension Rates of E.coli and Deinococcus RecA Along Duplex DNA

RecA is a protein which promotes the exchange between two homologous DNA molecules in homologous recombination process. When individual RecA molecules assemble on DNA, the DNA is stretched and underwound to form a nucleoprotein filament with its rigidity and end-to-end length increased. We have developed single-molecule tethered particle motion (TPM) experiments to study the assembly dynamics of RecA proteins on individual duplex DNA molecules. The TPM method is capable of measuring the changes in DNA length by observing the bead's Brownian motion, thus allowing us to monitor RecA nucleation and extension in real-time. Using much shorter DNA (a few hundreds basepairs), TPM experiments offer improved sensitivity, since the DNA length change can be readily detected as soon as a few RecA bounded to duplex DNA molecules. Our experiments indicated a faster nucleation rate compared to the previous reports (Galletto et al., 2006). Moreover, we have compared the nucleation and extension rates of E. coli RecA with the RecA from Deinococcus radiodurans, UV-resistant bacteria, under different nucleotide states, ATP and ATPγS. Deinococcus radiodurans RecA are found to nucleate faster (∼1.6×10-2 bp-1min-1) but extend slower (∼0.3-1.5 RecA/sec) under ATP.This difference reflects the physiological role of Dr. RecA when extensive UV-damaged DNA molecules are present.

Tetramer opening in LacI-mediated DNA looping

Lactose repressor protein (LacI) controls transcription of the genes involved in lactose metabolism in bacteria. Essential to optimal LacI-mediated regulation is its ability to bind simultaneously to two operators, forming a loop on the intervening DNA. Recently, several lines of evidence (both theoretical and experimental) have suggested various possible loop structures associated with different DNA binding topologies and LacI tetramer structural conformations (adopted by flexing about the C-terminal tetramerization domain). We address, specifically, the role of protein opening in loop formation by employing the single-molecule tethered particle motion method on LacI protein mutants chemically cross-linked at different positions along the cleft between the two dimers. Measurements on the wild-type and uncross-linked LacI mutants led to the observation of two distinct levels of short tether length, associated with two different DNA looping structures. Restricting conformational flexibility of the protein by chemical cross-linking induces pronounced effects. Crosslinking the dimers at the level of the N-terminal DNA binding head (E36C) completely suppresses looping, whereas cross-linking near the C-terminal tetramerization domain (Q231C) results in changes of looping geometry detected by the measured tether length distributions. These observations lead to the conclusion that tetramer opening plays a definite role in at least a subset of LacI/DNA loop conformations.

Probing The Structure And Function Of Transcription Complex Of RNA Polymerase II With TFIIF At Single Molecular Level

Previously, we have developed a bio-conjugation method that allows us to specifically label the C-terminus of any subunit of a RNA polymerase. Here, we reconstitute a RNA polymerase II (Pol II) with TFIIF (IIF), in which the C-terminus of a RNA polymerase subunit is labeled with Cy3 and an amino acid of the largest subunit of TFIIF is labeled with Cy5. By measuring FRET at single molecular level, we are able to probe the structure information of Pol II/IIF in the absence of X-ray structure. By using single molecular FRET and tethered particle motion method, we are also carrying out transcription of Pol II in the presence or absence of IIF to identify the role of IIF in RNA elongation at various stages.

Structure Conversion Of Human Telomeric Sequence Studied By Single-molecule Tethered Particle Motion

Telomeres contain G-rich tandem repeats of single-stranded DNA sequences at 3′ tail. The G-rich sequences can be folded into a secondary structure named G-quadruplexes by Hoogsteen base pairing in the presence of monovalent cations (such as Na+, K+). The folding of telomeric DNA into the G-quadruplexes may inhibit telomerase activity for the proliferation of cancer cells. Moreover, the change of a quadruplex conformation may play an important role in biological effect. Thus, understanding structure conversion between the folded and unfolded G-quadruplex structures, and how the structure conversion is mediated by ions, its anti-sense sequence and its stabilizers are important to telomere biology. Here, we have directly monitored the conversion between the folded and unfolded structures in human telomeric AGGG(TTAGGG)3 sequence by the single-molecule tethered particle motion (TPM) method. TPM method monitors the DNA length change caused by the G-quadruplex formation, and allows us to monitor the conversion mechanism of telomeric sequences in real-time. In the presence of its antisense sequence, the folded G-quadruplex structures (in 150 mM Na+) can be disrupted and converted to the unfolded conformation, and the conversion frequency depends strongly on the antisense concentrations. In the great excess of antisense sequence, the conversion efficiency is about 10 % in our single-molecule essay (N >100). However, in the presence of Li+ ions, the efficiency of antisense interaction increases significantly to 50 %. Since Li+ ions have been proposed to destabilize the G-quadruplex structure, our results suggest the antisence sequence interacts with the unfolded or, at least, partially unfolded state of telomeric sequences. Experiments of structure conversion between 150 mM Na+ and 100 mM K+ ions, and effects of structure conversion in the presence of a G-quadruplex stabilizer (BMVC) will also be discussed.

3P-098 TPM法を用いたタンパク質翻訳中におけるmRNAに沿ったリボソームの運動解析(核酸-相互作用・複合体,第47回日本生物物理学会年会)

3P-098 TPM法を用いたタンパク質翻訳中におけるmRNAに沿ったリボソームの運動解析(核酸-相互作用・複合体,第47回日本生物物理学会年会)

Analysis of Kinetics in Noisy Systems: Application to Single Molecule Tethered Particle Motion

In the tethered particle motion method the length of a DNA molecule is monitored by measuring the range of diffusion of a microsphere tethered to the surface of a microscope coverslip through the DNA molecule itself. Looping of DNA (induced by binding of a specific protein) can be detected with this method and the kinetics of the looping/unlooping processes can be measured at the single molecule level. The microsphere’s position variance represents the experimental variable reporting on the polymer length. Therefore, data windowing is required to obtain position variance from raw position data. Due to the characteristic diffusion time of the microsphere, the low-pass filtering required to attain a good signal/noise ratio (S/N) in the discrimination of looped versus unlooped state impacts significantly the measurement’s time resolution. Here we present a method for measuring lifetimes based on half-amplitude thresholding and then correcting the kinetic measurements, taking into account low S/N (leading to false events) and limited time resolution (leading to missed events). This method allows an accurate and unbiased estimation of the kinetic parameters under investigation, independently of the choice of the window used for variance calculation, with potential applications to other single molecule measurements with low S/N.

Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule

Schafer et al. (Nature 352:444-448 (1991)) devised the tethered particle motion (TPM) method to detect directly the movement of single, isolated molecules of a processive nucleic acid polymerase along a template DNA molecule. In TPM studies, the polymerase molecule is immobilized on a glass surface, and a particle (e.g., a 0.23 microns diameter polystyrene bead) is attached to one end of the enzyme-bound DNA molecule. Time-resolved measurements of the DNA contour length between the particle and the immobilized enzyme (the "tether length") are made by determining the magnitude of the Brownian motion of the DNA-tethered particle using light microscopy and digital image processing. We report here improved sample preparation methods that permit TPM data collection on transcript elongation by the Escherichia coli RNA polymerase at rates (approximately 10(2)-fold higher than those previously obtained) sufficient for practical use of microscopic kinetics techniques to analyze polymerase reaction mechanisms. In earlier TPM experiments, calculation of tether length from the observed Brownian motion was based on an untested numerical simulation of tethered bead Brownian motion. Using the improved methods, we have now empirically validated the TPM technique for tether lengths of 308-1915 base pairs (bp) using calibration specimens containing particles tethered by individual DNA molecules of known lengths. TPM analysis of such specimens yielded a linear calibration curve relating observed Brownian motion to tether length and allowed determination of the accuracy of the technique and measurement of how temporal bandwidth, tether length, and other experimental variables affect measurement precision. Under a standard set of experimental conditions (0.23 microns diameter bead, 0.23 Hz bandwidth, 23 degrees), accuracy is 108 and 258 bp r.m.s. at tether lengths of 308 and 1915 bp, respectively. Precision improves linearly with decreasing tether length to an extrapolated instrumentation limit of 10 bp r.m.s. and improves proportionally to the inverse square root of measurement bandwidth (1.9 x 10(2) bp Hz-1/2 for 1090-bp tethers). Measurements on large numbers of individual polymerase molecules reveal that time-averaged single-molecule elongation rates are more variable than is predicted from the random error in TPM measurements, demonstrating that the surface-immobilized RNA polymerase molecules are kinetically heterogeneous.