Bulletin of the American Physical Society
2006 APS March Meeting
Monday–Friday, March 13–17, 2006; Baltimore, MD
Session D26: Focus Session: Dynamics of Nuclei Acid-Protein Interaction: Single Molecule |
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Sponsoring Units: DBP DPOLY Chair: Mark C. WiIliams, Northeastern University Room: Baltimore Convention Center 323 |
Monday, March 13, 2006 2:30PM - 3:06PM |
D26.00001: Probing Nucleosome Remodeling by Unzipping Single DNA Molecules Invited Speaker: At the core of eukaryotic chromatin is the nucleosome, which consists of 147 bp of DNA wrapped 1.65 turns around an octamer of histone proteins. Even this lowest level of genomic compaction presents a strong barrier to DNA-binding cellular factors that are required for essential processes such as transcription, DNA replication, recombination and repair. Chromatin remodeling enzymes use the energy of ATP hydrolysis to regulate accessibility of the genetic code by altering chromatin structure. While remodeling enzymes have been the subject of extensive research in recent years, their precise mechanism remains unclear. In order to probe the structure of individual nucleosomes and their remodeling, we assembled a histone octamer onto a DNA segment containing a strong nucleosome positioning sequence. As the DNA double helix was unzipped through the nucleosome using a feedback-enhanced optical trap, the presence of the nucleosome was detected as a series of dramatic increases in the tension in the DNA, followed by sudden tension reductions. Analysis of the unzipping force throughout the disruption accurately revealed the spatial location and fine structure of the nucleosome to near base pair precision. Using this approach, we investigate how remodeling enzymes may alter the location and structure of a nucleosome. [Preview Abstract] |
Monday, March 13, 2006 3:06PM - 3:18PM |
D26.00002: Binding Study of T7 Gene 2.5 Protein to Single- and Double--Stranded DNA from Single Molecule Stretching Leila Shokri, Boriana Marintcheva, Charles C. Richardson, Mark C. Williams Bacteriophage T7 gene 2.5 protein binds preferentially to single-stranded DNA. This property is essential for its role in DNA replication, recombination, and repair. We present the first quantitative study of the thermodynamics and kinetics of equilibrium and non-equilibrium DNA helix destabilization in the presence of gp2.5 and a deletion mutant lacking 26 C-terminal amino acids that binds with higher affinity to ssDNA (gp2.5-delta26C). Our measured force-extension curves of lambda-DNA in the presence of these proteins suggest strong cooperative binding. By measuring the DNA melting force as a function of time and pulling rate, we obtained binding site size and the association constants of these proteins to ssDNA and dsDNA, over a range of salt and protein concentrations. The results are used to characterize the electrostatic interactions that determine the DNA-protein binding in each case. [Preview Abstract] |
Monday, March 13, 2006 3:18PM - 3:30PM |
D26.00003: Dynamics of Protein-DNA Interactions probed with Laser Temperature-Jump and Time-Resolved FRET Measurements. Serguei Kuznetsov, Paula Vivas, Sawako Sugimura, Donald Crothers, Anjum Ansari In many protein-DNA complexes, the DNA is often bent or sharply kinked. In order to elucidate the energetics of the binding mechanism it is necessary to study the dynamics of the binding and bending of DNA. Previous kinetics measurements failed to resolve DNA bending on the millisecond time-scales of stopped-flow techniques. Here we report measurements on the binding of IHF, an architectural protein from \textit{E. coli}, to its native H' substrate end-labeled with FRET pair to monitor the DNA bending. Stopped-flow measurements show relaxation kinetics that become concentration independent at high IHF concentrations, suggesting that under these conditions, the DNA bending becomes rate-limiting. To test this interpretation, we use a laser temperature-jump to perturb the IHF-H' complex, and to probe the dynamics with submillisecond time-resolution. These measurements support a sequential model for DNA binding and bending to IHF. The time-scales for DNA bending, when in complex with the protein, are not inconsistent with thermal fluctuations that can spontaneously bend DNA. [Preview Abstract] |
Monday, March 13, 2006 3:30PM - 3:42PM |
D26.00004: Inferring the \textit{in vivo} looping properties of DNA. Jose Vilar, Leonor Saiz, Miguel Rubi The free energy of looping DNA by proteins and protein complexes determines to what extent distal DNA sites can affect each other [1]. We inferred its \textit{in vivo} value through a combined computational-experimental approach for different lengths of the loop [2] and found that, in addition to the intrinsic periodicity of the DNA double helix, the free energy has an oscillatory component with about half the helical period. Moreover, the oscillations have such an amplitude that the effects of regulatory molecules become strongly dependent on their precise DNA positioning and yet easily tunable by their cooperative interactions. These unexpected results can confer to the physical properties of DNA a more prominent role at shaping the properties of gene regulation than previously thought. [1] J.M.G. Vilar and L. Saiz, Current Opinion in Genetics {\&} Development, 15, 136-144 (2005). [2] L. Saiz, J.M. Rubi, and J.M.G. Vilar, Proc. Natl. Acad. Sci. USA, 102, 17642-17645 (2005). [Preview Abstract] |
Monday, March 13, 2006 3:42PM - 4:18PM |
D26.00005: DNA kept under tension reveals mechanochemical properties of protein reaction pathways Invited Speaker: The genetic information of an organism is encoded in the base pair sequence of its DNA. Many specialized proteins are involved in handling DNA, preserving and processing the vast amounts of information on the DNA. In order to do this swiftly and correctly these proteins have to move quickly and accurately along and/or around the DNA. Using model systems such as restriction enzymes and abundant bacterial gene regulators such as H-NS we try to understand the physics (forces, energies, mechanochemistry) behind such DNA processing. We are currently performing single molecule experiments on (non-)specific protein-DNA interactions in general and the organization of the bacterial nucleoid in particular. The experiments aim to elucidate the induced-fit reaction, substrate binding and DNA hydrolysis. Moreover, we are studying the relation between DNA configuration and association rates. The results of these model systems are generalized and though to be applicable to many DNA-protein interactions. [Preview Abstract] |
Monday, March 13, 2006 4:18PM - 4:30PM |
D26.00006: A Model for Folding and Aggregation in RNA Secondary Structures Vishwesha Guttal, Ralf Bundschuh We study the statistical mechanics of RNA secondary structures designed to have an attraction between two different types of structures as a model system for heteropolymer aggregation. The competition between the branching entropy of the secondary structure and the energy gained by pairing drives the RNA to undergo a `{\it temperature independent}' second order phase transition from a molten to an {\it aggregated phase}. The aggregated phase thus obtained has a macroscopically large number of contacts between different RNAs. The partition function scaling exponent for this phase is $\theta \approx 1/2$ and the crossover exponent of the phase transition is $\nu \approx 5/3$. The relevance of these calculations to the aggregation of biological molecules is discussed. [Preview Abstract] |
Monday, March 13, 2006 4:30PM - 4:42PM |
D26.00007: Mechanism of gene-regulating protein’s diffusion along DNA: hopping vs. sliding Yan Mei Wang, Edward Cox, Robert Austin It has been a long controversy as whether non-energy-driven proteins diffuse along DNA in the form of hopping or sliding. In the hopping model, the protein jumps on and off DNA frequently while diffusing, switching between 1D and 3D diffusions, and in the sliding model the protein diffuses along DNA basepair by basepair, staying in continuous contact with the DNA. We have investigated the diffusion mechanisms of LacI repressor protein along nonspecific sequences of DNA using single molecule imaging measurements. By studying the standard deviation (SD) of the diffusing LacI’s point spread functions, we observed that the SD values in both the longitudinal and transverse directions to DNA elongation to be significantly higher than what can be accounted for by the sliding model. We will show that the large SD values agree with the hopping model. [Preview Abstract] |
Monday, March 13, 2006 4:42PM - 4:54PM |
D26.00008: Protein jamming on DNA Zeba Wunderlich, Michael Slutsky, Mehran Kardar, Leonid Mirny The mechanisms by which DNA-binding proteins find their sites on DNA have been the subject of intensive research in biophysics. Most theoretical models consider proteins searching naked DNA for their binding sites. In the cell, however, myriads of proteins and protein complexes ($e.g.$ nucleosomes) are constantly binding DNA. What is the effect of DNA-bound proteins and nucleosomes on the rate of protein-DNA association and dissociation? Here we study how a protein finds its site on DNA, and how long it stays on its site, considering DNA that is bound by other proteins. The process of association and dissociation is modeled by rounds of 3D diffusion and 1D sliding. We assume that proteins bound to DNA act as reflecting boundaries and obstruct sliding of other proteins. We demonstrate that if the density of proteins on the DNA is above a critical level, a protein's mobility on DNA is significantly reduced, leading to jamming. We find that proteins bound to DNA in the proximity of the specific site can (i) significantly increase the time it takes for the protein to find its site, and (ii) simultaneously increase the time a protein spends on its site. The increase in the residence time of a protein on its site can have important biological implications. Our results are consistent with recent experiments on p53 and the organization of nucleosomes in yeast promoters. The structures of eukaryotic enhancers also suggest that jamming may play a role in the assembly of protein complexes on DNA. [Preview Abstract] |
Monday, March 13, 2006 4:54PM - 5:06PM |
D26.00009: Single-molecule Study of Nucleocapsid Protein Chaperoned DNA Hairpin Structural Dynamics Yining Zeng, Gonzalo Cosa, Hsiao-Wei Liu, Christy Landes, Dmitrii Makarov, Paul Barbara, Karin Musier-Forsyth In HIV-1 reverse transcription, the nucleocapsid protein, NC, induces secondary structure fluctuations in the transactivation response (TAR) region of DNA and RNA hairpins. Time resolved single-molecule fluorescence resonance energy transfer was used to study NC chaperoned secondary fluctuations of DNA hairpins. The experiments reveal that the NC induced secondary fluctuations are limited to the terminal stems, and the mechanism for the fluctuations is complex. The dynamic processes occur over a wide time range, i.e. $\sim $5 to $>$250 milliseconds and involve long-lived intermediates. The dynamic role of DNA loop regions and NC binding/dissociation events are discussed. [Preview Abstract] |
Monday, March 13, 2006 5:06PM - 5:18PM |
D26.00010: Mechanism of Nucleic Acid Chaperone Function of Retroviral Nuceleocapsid (NC) Proteins Ioulia Rouzina, My-Nuong Vo, Kristen Stewart, Karin Musier-Forsyth, Margareta Cruceanu, Mark Williams Recent studies have highlighted two main activities of HIV-1 NC protein contributing to its function as a universal nucleic acid chaperone. Firstly, it is the ability of NC to weakly destabilize all nucleic acid,(NA), secondary structures, thus resolving the kinetic traps for NA refolding, while leaving the annealed state stable. Secondly, it is the ability of NC to aggregate NA, facilitating the nucleation step of bi-molecular annealing by increasing the local NA concentration. In this work we use single molecule DNA stretching and gel-based annealing assays to characterize these two chaperone activities of NC by using various HIV-1 NC mutants and several other retroviral NC proteins. Our results suggest that two NC functions are associated with its zinc fingers and cationic residues, respectively. NC proteins from other retroviruses have similar activities, although expressed to a different degree. Thus, NA aggregating ability improves, and NA duplex destabilizing activity decreases in the sequence: MLV NC, HIV NC, RSV NC. In contrast, HTLV NC protein works very differently from other NC proteins, and similarly to typical single stranded NA binding proteins. These features of retroviral NCs co-evolved with the structure of their genomes. [Preview Abstract] |
Monday, March 13, 2006 5:18PM - 5:30PM |
D26.00011: Fis protein induced $\lambda $F-DNA bending observed by single-pair fluorescence resonance energy transfer Fu Chi-Cheng, Fann Wunshain , Yuan Hanna S. Fis, a site-specific DNA binding protein, regulates many biological processes including recombination, transcription, and replication in E.coli. Fis induced DNA bending plays an important role in regulating these functions and bending angle range from $\sim $50\r{ } to 95 \r{ } dependent on the DNA sequence. For instance, the average bending angle of $\lambda $F-DNA (26 bp, 8.8nm long, contained $\lambda $F binding site on the center) measured by gel mobility shift assays was $\sim $ 94 \r{ }. But the traditional method cannot provide information about the dynamics and the angle distribution. In this study, $\lambda $F-DNA was labeled with donor (Alexa Fluor 546) and acceptor (Alexa Fluor 647) dyes on its two 5' ends and the donor-acceptor distances were measured using single-pair fluorescence resonance energy transfer (sp-FRET) with and without the present of Fis protein. Combing with structure information of Fis-DNA complex, the sp-FRET results are used to estimate the protein induced DNA bending angle distribution and dynamics. [Preview Abstract] |
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