83 research outputs found
Mechanical response of plectonemic DNA: an analytical solution
We consider an elastic rod model for twisted DNA in the plectonemic regime.
The molecule is treated as an impenetrable tube with an effective, adjustable
radius. The model is solved analytically and we derive formulas for the contact
pressure, twisting moment and geometrical parameters of the supercoiled region.
We apply our model to magnetic tweezer experiments of a DNA molecule subjected
to a tensile force and a torque, and extract mechanical and geometrical
quantities from the linear part of the experimental response curve. These
reconstructed values are derived in a self-contained manner, and are found to
be consistent with those available in the literature.Comment: 14 pages, 4 figure
Reconstruction of bacterial transcription-coupled repair at single-molecule resolution
International audienc
Elasticity and electrostatics of plectonemic DNA
We present a self-contained theory for the mechanical response of DNA in
single molecule experiments. Our model is based on a 1D continuum description
of the DNA molecule and accounts both for its elasticity and for DNA-DNA
electrostatic interactions. We consider the classical loading geometry used in
experiments where one end of the molecule is attached to a substrate and the
other one is pulled by a tensile force and twisted by a given number of turns.
We focus on configurations relevant to the limit of a large number of turns,
which are made up of two phases, one with linear DNA and the other one with
superhelical DNA. The model takes into account thermal fluctuations in the
linear phase and electrostatic interactions in the superhelical phase. The
values of the torsional stress, of the supercoiling radius and angle, and key
features of the experimental extension-rotation curves, namely the slope of the
linear region and thermal buckling threshold, are predicted. They are found in
good agreement with experimental data.Comment: 19 pages and 6 figure
Diffusion of transcription factors can drastically enhance the noise in gene expression
We study by simulation the effect of the diffusive motion of repressor
molecules on the noise in mRNA and protein levels in the case of a repressed
gene. We find that spatial fluctuations due to diffusion can drastically
enhance the noise in gene expression. For a fixed repressor strength, the noise
due to diffusion can be minimized by increasing the number of repressors or by
decreasing the rate of the open complex formation. We also show that the effect
of spatial fluctuations can be well described by a two-step kinetic scheme,
where formation of an encounter complex by diffusion and the subsequent
association reaction are treated separately. Our results also emphasize that
power spectra are a highly useful tool for studying the propagation of noise
through the different stages of gene expression.Comment: 15 pages, 6 figures, REVTeX
Efficient preparation of internally modified single-molecule constructs using nicking enzymes
Investigations of enzymes involved in DNA metabolism have strongly benefited from the establishment of single molecule techniques. These experiments frequently require elaborate DNA substrates, which carry chemical labels or nucleic acid tertiary structures. Preparing such constructs often represents a technical challenge: long modified DNA molecules are usually produced via multi-step processes, involving low efficiency intermolecular ligations of several fragments. Here, we show how long stretches of DNA (>50 bp) can be modified using nicking enzymes to produce complex DNA constructs. Multiple different chemical and structural modifications can be placed internally along DNA, in a specific and precise manner. Furthermore, the nicks created can be resealed efficiently yielding intact molecules, whose mechanical properties are preserved. Additionally, the same strategy is applied to obtain long single-strand overhangs subsequently used for efficient ligation of ss- to dsDNA molecules. This technique offers promise for a wide range of applications, in particular single-molecule experiments, where frequently multiple internal DNA modifications are required
The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex.
We incorporated the non-natural photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa) into the RNA polymerase II (Pol II) surface surrounding the central cleft formed by the Rpb1 and Rpb2 subunits. Photo-cross-linking of preinitiation complexes (PICs) with these Pol II derivatives and hydroxyl-radical cleavage assays revealed that the TFIIF dimerization domain interacts with the Rpb2 lobe and protrusion domains adjacent to Rpb9, while TFIIE cross-links to the Rpb1 clamp domain on the opposite side of the Pol II central cleft. Mutations in the Rpb2 lobe and protrusion domains alter both Pol II-TFIIF binding and the transcription start site, a phenotype associated with mutations in TFIIF, Rpb9 and TFIIB. Together with previous biochemical and structural studies, these findings illuminate the structural organization of the PIC and the network of protein-protein interactions involved in transcription start site selection
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