Biology Reference
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rotation of the trapped bead. These data have provided strong evidence that
sliding is used by RNAP. In another study, Yanagida and coworkers 50 sus-
pended a single extended DNA filament between two trapped beads and
found that RNAP performed a randomwalk along the DNA. Upon release of
the DNA tension, the DNA reformed a random coil conformation and it was
noted that the proteins bound to theDNA targets with greater efficiency. This
could imply either that DNA tension is preventing efficient target location or
that the proteins bind and release rapidly and that their redistribution to the
promoter sites is favored by the formation of a compact DNA structure that
allows the proteins to three-dimensionally hop between sites on the DNA, as
suggested by a later study using restriction endonucleases. 73 The use of laser
tweezers has permitted a number of elegant studies on the single-molecule
properties of DNA-protein interactions, 74-78 including obtaining base pair
resolution of RNAP stepping. 79 Despite these advances, optical trapping is
not a trivial technique to apply as it requires specialist equipment and
expertise. In addition, data collection is not multiplexed and is therefore
slow as each investigation typically provides information on a single DNA
strand. However, the recent use of multichannel fluidics could overcome
some of the latter shortcomings. 47
2. Magnetic tweezers offer an approach that harnesses much of the power
of laser tweezers with the potential for more multiplexing. This approach
has been used to investigate the function of a number of enzymes bound to
DNA that alter its end-to-end length. Such length variations occur through
alterations in single-stranded and double-stranded character 80 or
topoisomerase-induced relaxation of DNA, 81 among others. For magnetic
tweezers to provide detailed information on the physical basis of target site
location, however, further development to combine this with fluorescence
detection is required.
3. Direct imaging is currently in a position to provide direct information on
the mechanism of target site acquisition for DNA-binding proteins. To
date, a number of studies have provided diffusion constants and hopping
distances (many summarized in Table I ). These studies have invariably
required the extension of the DNA substrate from its random coil to
facilitate imaging of protein motions. To achieve this, DNA can be laid
directly down on a surface by combing. 82,83 This approach allows the
highest signal to noise ratio because imaging can be performed in the
evanescent field of a total internal reflection microscope (TIRF-equipped
microscope). Typically, the surface is activated with a hydrophobic moiety
such as polystyrene or polymethylmethacrylate. 82 At low pH, DNA binds to
these surfaces at two or more points; indeed, the number of binding points
can be visualized by snapping the DNA under intense illumination, at
which time the DNA recoils to its bound points on the surface. 83 This
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