Experiments in Physical Biology

Introductory tutorial on the basics of DNA by John H. Maddocks - April 28, 2005, 10:10 am-12:00 pm, Lind Hall 409
Slides:   pdf

This is a strictly elementary introduction to the basics of DNA, aimed at mathematicians with no background in biochemistry, with the objective of establishing a rudimentary foundation for Part I of the workshop "Experiments in Physical Biology," May 2-6.

Zev Bryant (Department of Biochemistry, Stanford University)

Single molecule mechanics of DNA

I will begin with a review of single molecule manipulation techniques applied to DNA and what they have taught us about DNA physics and enzymology. I will then concentrate on direct measurements of torque and twist in single stretched DNA molecules. Torque and twist measurements have provided insights into the mechanical properties of DNA and laid the experimental and conceptual groundwork for a new class of mechanistic studies of DNA-associated enzymes. I will conclude with an example of an enzymogical application: a mechanochemical dissection of E. Coli DNA gyrase.

Nikolaus P. Ernsting (Department of Chemistry, Humboldt University, Berlin)

Molecular probes for DNA structure and dynamics
Slides:  Part I       Part II

Charge separation and subsequent transport along the pi-stack is one of the mechanisms of radiative damage in double-stranded DNA. Charge transfer (CT) is controlled by electronic interaction between nearby nucleobases, and these interactions in turn depend on the local structure and its fluctuation on a picosecond time scale. The approach up to now has been to measure the charge transfer rate for well-devised systems. In the first lecture I will introduce the basic concepts and outline the most influential experiments to date. Here we aim for an elementary understanding of the distance or sequence dependence of CT. Recent experiments which demonstrate gating of charge transfer by the motion of nucleobases or solvent will also be discussed.

The second lecture focuses on structure fluctuations, local (1-3 basepairs) and fast (100 fs- 10 ps), but addresses them separate from a CT process. For this purpose one needs suitable molecular probes. Their excitation with femtosecond light pulses perturbs the structure, and their fluorescence evolution reports the response. The status, scope, and analysis needs of femtosecond bandshape measurements will be discussed.

(1) Charge Transfer in ds DNA

Energetics and Electronics of Charge Transfer
Charge injection: photochemical approach
Charge injection: photophysical approach
Reason for distance dependence
Conformational dynamics influencing Charge Transfer

Motions and distributions of nucleobases
Solvation dynamics

Model for the dye in its environment
Spectral evolution
Example using broadband transient absorption:   local charge fluctuations
Example using broadband fluorescence upconversion: free volume

Nicholas Hud (Georgia Institute of Technology)

DNA condensation

Free DNA in solution exists in a quasi-extended state that is often modeled as a worm-like chain. However, in living cells DNA exists in much more compact and ordered states. The most highly compact forms of DNA are those found in viruses and sperm cells, where compaction is of paramount importance and immediate access to the genetic code is not required. In the laboratory, multivalent cations can be used to condense DNA from solution into nanometer-scale particles with morphologies that resemble DNA packaged in viruses and sperm cells. Experimental investigations of DNA condensation in vitro have already revealed much about the physical properties of DNA. Continuing studies of DNA condensation by proteins isolated from different cell types are providing new insights into how high-density packing of DNA is achieved by living organisms.

Jason D. Kahn (Department of Chemistry and Biochemistry, University of Maryland) http://www.biochem.umd.edu/biochem/kahn/

DNA bending, twisting, and looping in the control of transcription

Proteins that activate or repress gene expression often bring about DNA deformation, and consequently they respond to DNA shape and flexibility. Examples include the TATA box binding protein (TBP) central to eukaryotic transcription, the lactose repressor (LacI), and the NtrC transcriptional activator . DNA cyclization kinetics applied to the TBP-DNA complex demonstrates unusual anisotropic flexibility of the unbound TATA box DNA sequence and also confirms the general DNA bend observed in crystal structures. The surprising appearance of negatively supercoiled products upon ring closure of short bound DNA suggests that the TBP-DNA complex is capable of flattening in response to external stress, which may be relevant to the recruitment of TBP to remodeled chromatin. Electrophoretic mobility shift, cyclization, Monte Carlo simulation, and bulk and single-molecule FRET experiments applied to DNA constructs designed to stabilize different LacI-DNA loop geometries demonstrate that the protein can adopt both the V-shaped form seen in crystal structures and also a more open conformation. The choice between geometries is dictated by both protein and DNA shape and deformability. Finally, In vivo results on transcriptional activation of E. coli sigma-54 RNA polymerase by NtrC suggest that protein multivalency and flexibility abrogate the expected influence of DNA shape, but suggest that dynamic DNA wrapping within an NtrC enhanceosome may be essential to its function.

Sarah L. Keller (Department of Chemistry, University of Washington) http://depts.washington.edu/chemfac/keller.html

Abstract for the 2 talks:
(1) A gentle introduction to lipid membranes and resulting miscibility phase diagrams
(2) Seeing spots: experimentally determining lipid miscibility phase diagrams in free-floating vesicles, supported membranes, and monolayers at an air-water interface

Mammalian cells are surrounded by an outer wall or "plasma membrane" of proteins and lipids arranged in opposing leaflets of a bilayer. There is growing evidence that this membrane is not uniform, but instead laterally phase separates into "raft" domains rich in particular lipids and proteins. We study a simpler physical model of cell membranes, giant unilamellar vesicles (GUVs). Liquid domains in vesicles exhibit interesting behavior: they collide and coalesce, can finger into stripes, and can bulge out of the vesicle. We use fluorescence microscopy to directly observe liquid domains in the vesicles. We cross miscibility phase transitions by changing temperature. Using results from both fluorescence microscopy and NMR studies, we quantitatively construct tie-lines on phase diagrams. These tie-lines allow us to estimate free energies to transfer lipids between phases. We also find that it is possible to capture domains in lipid layers on glass substrates.

Thomas D. Pollard (Departments of Molecular, Cellular & Developmental Biology, of Molecular Biophysics and Biochemistry and of Cell Biology, Yale University, New Haven CT)

Quantitative analysis of actin-based cellular motility and cytokinesis
Review:  pdf

My research group uses a combination of biophysical methods, molecular genetics and microscopy to study how cells assemble actin filaments to produce forces for cellular motility and cytokinesis. Recently we developed methods to measure the global and local concentrations of many of the key proteins in live cells.

Arp2/3 complex nucleates a network of branched actin filaments that pushes forward the leading edge of motile eukaryotic cells and cortical actin patches in fungi. We propose that activation of Arp2/3 complex involves a conformational change that juxtaposes Arp2 and Arp3 and that the active conformation is stabilized by cooperative binding of WASp/Scar nucleation promoting factors, the first actin subunit in the daughter filament and mother filaments. Rho-family GTPases and membrane polyphosphoinositides activate WASp, presumably on the inner surface of the plasma membrane. Some of these events can be visualized in real time in fission yeast actin patches.

Formins are a second class of GTPase-activated actin filament nucleation proteins. Cdc12p, the formin required for cytokinesis of fission yeast, is a profilin-gated barbed end capping protein. Single molecule experiments show that Cdc12p and budding yeast Bni1p remain bound to an elongating barbed ends for >1000 seconds and that polymerization produces picoNewton forces. Five different formins all bind to the barbed end of actin filaments but vary in their capping activity. Profilin increases the rate of formin-mediated addition both ATP- and ADP-actin monomers to barbed ends, so ATP hydrolysis is not required for this process.

M. Thomas Record, Jr. (Department of Chemistry, University of Wisconsin)

Wrapping of DNA on protein surfaces

M. Thomas Record, Jr. (Department of Chemistry, University of Wisconsin)

Large-scale conformational changes in RNA polymerase and promoter DNA in transcription initiation

Opening of the transcription start site and the adjacent regions of promoter DNA by RNA polymerase is accomplished thermodynamically using binding free energy. The mechanism of binding to and opening the promoter involves a sequence of large scale conformational changes. Bending and wrapping of the upstream DNA occur early in the process; coupled folding of regions of polymerase occurs late, and the kinetics are dominated by a slow conformational change once the duplex DNA is in the jaw-like structure of the polymerase. Kinetic and footprinting studies to eluciate the nature of this conformational change and of the intermediates will be discussed.

Robert Schleif (Department of Biology, Johns Hopkins University)

DNA looping: biology and mechanisms

What it is.
Why Nature uses it.
How it was discovered.
Additional experiments demonstrating looping.
Role of supercoiling in looping.
How looping is controlled in the arabinose system.

Jonathan Widom (Department of Biochemistry, Molecular Biology and Cell Biology and Department of Chemistry, Northwestern University)

Sequence-dependent DNA mechanics in very small DNA circles and nucleosomes

Part I of my lectures will summarize our recent studies on the spontaneous bending and twisting of double stranded DNA. We found that DNAs that are much shorter than the DNA persistence length easily form very small circles, whose inner diameters are only a few times larger than the diameter of the DNA from which they are formed. I will discuss our experiments that demonstrate that these small circles exist, summarize our current understanding of the bendability and twistability of the sharply bent DNA, and discuss striking effects of particular DNA sequences on the relative ability of the circles to form. Part II will focus on sharply bent DNA as it occurs in nucleosomes, the fundamental structural subunits of chromosomes in people and all other higher life forms. I will discuss a selection experiment for DNA sequences that make especially stable nucleosomes, unexpected sequence motifs discovered in these selected DNAs that are responsible for their facile nucleosome formation, the relation between nucleosome formation and spontaneous DNA cyclization, and current ideas for the molecular basis of sequence dependent DNA bendability.