Chemistry Reference
In-Depth Information
Figure 9.10 ALEX-FRET on single diffusing
complexes allows simultaneous monitoring of
movement of RNAP on DNA and association of
transcription factor
smaller D
A distance), and S is the
stoichiometric ratio (S
-
¼
1 for D-only, S
¼
0 for
A-only, and S
0.5 for both D and A present).
(A) Subpopulations identified using ALEX-FRET
may be followed as a reaction progresses.
Reproduced with permission from [95].
¼
70
with RNAP core. Two-
dimensional E-S histograms of single molecule
fluorescence bursts are shown. E is the FRET
efficiency (low for larger D
s
A distance, high for
-
RNAP was attached to the surface, and the DNA was not. Initial encouraging results
showing activity were found to be photophysical in origin. At the time we were unable
to determine whether RNAP was functioning properly on the surface. An additional
problem was that the single-molecule FRETmethodology, as then constituted, was
unable to tell the difference between a molecule with only the donor attached and a
molecule with the donor bound to amolecule with the acceptor, but too far away to see
