Biology Reference
In-Depth Information
movement in any one direction. Thus, directed motion in which the protein
migrates in only one direction requires an input of energy. It is possible that the
ATPase sites in UvrA and UvrB could provide directed motion.
In another example of how UvrA
2
B
2
could search the genome, it has been
noted that this complex can, in principle, bind multiple DNA helices
56
and,
therefore, it offers a mechanism for transfer from one DNA region to another
without ever dissociating in what is called intersegmental transfer (see
Fig. 2
).
In this mode of searching, two DNA molecules would have to make close
contact. Such mechanisms of protein movement on DNA have been proposed
for restriction endonucleases and for lac repressor.
57,58
Clearly, there are a number of proposed mechanisms by which proteins can
negotiate DNA to find their target sites; however, definitive evidence for one
mechanism versus another is extremely difficult to obtain using bulk methods.
Therefore, more recently, there has been an explosion of studies using a variety of
single-molecule techniques that permit each individual protein complex to be
followed with good time and spatial resolution.
40,43,45,59-69
These studies are likely
to provide a clear understanding of which method(s) of protein transfer occurs.
Understanding the precise mechanism of how proteins locate their target sites is
of crucial importance and is discussed in more detail below.
C. Necessary Experimental Components to Observe
Single Molecules in Action
The original presentation of the lac repressor finding its target site faster
than the Smoluchowski-defined diffusional limit
70,71
was the first experimental
demonstration that the search mechanism may involve a 1D component in-
stead of being purely 3D. This subsequently prompted a number of papers
exploring the theoretical aspects of this potential mode of target location (see
Ref.
55
). In addition, a number of experimental studies have provided evidence
for and against these models which are summarized below. Single-molecule
techniques offer a direct method of determining how proteins find their targets
and have more recently been deployed in a number of experimental formats.
A few of these are summarized here:
1. Optical tweezers: By using a focused (normally infrared) beam, it is
possible to exert forces on dielectric spheres, thus ''trapping'' them at the
waist of the incident laser. This approach allows for the position of the trapped
bead to be manipulated in three dimensions, and by attaching a single strand
of DNA to the bead, the DNA is likewise manipulated. In an ingenious
experiment where one end of DNA was tethered to a bead and the other to
RNAP attached to a surface, it was shown that RNAP scans the groove of
DNA in search of its promoter
72
because movement of the surface led to
