Chemistry Reference
In-Depth Information
these methods is that only one channel is monitored, preventing the use of
ratiometric variables that are so bene
cial in FRET measurements. However, for
monitoring such short distances at the single-molecule level, there is no current
alternative to designing these more challenging experiments.
9.4.2
Single-molecule Measurements of DNA-processing Enzymes
The methodologies we have discussed in this chapter so far have been mainly
applicable to DNA
-
protein interactions rather than protein
-
protein interactions.
One reason is their importance: maintaining the
fidelity of DNA replication is
necessary for cell survival, and transcription is a primary point at which gene
expression is regulated. Another reason is practical: these methods require the
labeling of twomacromolecules in order to observe bothmovement and interactions.
Compared to proteins, DNA is relatively easy to label site speci
cally. Having DNA as
one of the labeled macromolecules lessens the work involved in getting the bio-
chemical system working.
We focus this section on the example of our measurements on RNA polymerase.
The methods can be generalized to other DNA-processing enzymes. Transcription is
the process of copying a gene from a DNA template to RNA, which in turn serves as
the template for protein synthesis. It is one of the main steps used in regulating the
expression levels of proteins, and thus maintaining the proper level of protein. RNA
polymerase (RNAP) is the protein machine that copies DNA into RNA. In collabora-
tion with the Ebright group, we set out to investigate the initiation of transcription.
Our initial measurements of RNAP were attempted with a surface-immobilized
format. Thesemeasurements proved to be an exercise in frustration. A donor-labeled
RNAP was supposed to bind to an acceptor-labeled DNA, and, upon adding the
required nucleotides, transcription would proceed in a controlled manner. The
Figure 9.9 (Continued )
environment (e.g. diffusion within the vesicle).
Two types of fluctuations are visible in the
E-trajectory: fast, step-wise fluctuations that
cannot be resolved and slow, continuous
transitions that can take
suggested by the bimodal FRET-efficiency
distributions. The landscape of AK is
characterized by local free energy barriers and
traps, resulting in fluorescence time-trajectories
that can start and end at any FRET-efficiency
value (the population weight of the folded
species is indicated by green bars, while the
unfolded conformers are color coded red). The
landscape of CI2 is smoother, and time
trajectories exhibit fluctuation between two
relatively constant levels of FRET-efficiencies. A
reproduced with permission from [87].
(Copyright 2004 American Chemical Society).
C reproduced with permission from [8].
(Copyright
1 s to complete. The
middle panel depicts a FRET-efficiency trajectory
calculated from the signals in the top panel,
whereas the interprobe distance trajectory
(calculated according to Forster theory) is shown
in the bottom panel. (C) Schematic of a one-
dimensional energy landscape for AK and CI2 at
denaturant concentrations close to the midpoint
of folding, obtained by averaging of the folding
landscape over many degrees of freedom and
projection onto the FRET-efficiency axis. Both
energy landscapes exhibit two global free energy
minima separated by a free energy barrier, as
>
2002 National Academy of
Sciences USA).
