Principles of Fluorescence Spectroscopy

460 ENERGY TRANSFER
Figure 13.24. RET (left) and Rac–GFP intensity (near 510 nm) image
(right) of motile 3T3 fibroblasts. Bar = 24 µm. Reprinted from [47].
single-stranded structure. Some RNA molecules are catalytically
active, which are called ribozymes. RET has found
use in studies of ribozyme folding and dynamics. 50–52
Figure 13.25 shows the donor, acceptor, sequence, and
secondary structure of the labeled hairpin ribozyme containing
the covalently linked donor and acceptor molecules.
52 After the substrate (S) binds it is cleaved by the
ribozyme. Cleavage requires binding of the A and B loops
to each other, a motion called docking. This motion is
expected to bring the donor and acceptor closer together
(lower panel). Upon addition of substrate there are changes
in both the donor and acceptor intensities (Figure 13.26).
The donor intensity decreases as expected for increased
energy transfer. The behavior of the acceptor is more complicated.
The acceptor intensity increases as expected, but
only after an initial drop in intensity on substrate binding.
This decrease is due to quenching of the acceptor by the
nearby guanine residue (Chapter 8).
13.6.1. Imaging of Intracellular RNA
The localization and concentration of intracellular mRNA
is an important factor in the control of protein synthesis.
Figure 13.25. Structure of the hairpin ribozyme labeled with donor
and acceptor. The lower panel shows the docking conformational
change that occurs upon binding of the substrate. Revised from [52].
Figure 13.26. Intensity changes of the donor and acceptor on the hairpin
ribozyme upon binding of substrate. Courtesy of Dr. Nils G.
Walter, University of Michigan.