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core enzyme), even as it was synthesizing the
9
-
11 nucleotide abortive RNA
transcripts. Three models were proposed to explain these observations. In the
rst
model, the DNA is pulled into the RNAP during initiation of transcription,
scrunching the DNA (model 1). In the second model, there is a
flexible element
in the RNAP that needs to stretch to another part of DNA in order to continue
transcription (inchworming). The third model explains the footprinting results
dynamically. The RNAP transientlymoves downstreamon theDNAuntil the abortive
transcript releases, and then backs up once the abortive transcript is released,
spending more time at the retracted position (Figure 9.11).
By using single-molecule leading- and trailing-edge FRET ALEX experiments on
freely-diffusingmolecules, we have been able to determine conclusively that theDNA
scrunching model is correct [1]. The single-molecule methods were able to obtain
these results in a simple, direct manner.
Similar solution-based measurements of DNA
-
protein interactions were per-
formed on HIV reverse transcriptase (with
fluorescence lifetime information, but
no ALEX), revealing heterogeneity in the reverse transcriptase/primer
-
template
complexes [43]. These measurements demonstrate the use of
uorescence
lifetime information to obtain structural
information regarding DNA
-
protein
interactions.
Observation of abortive initiation with immobilized molecule
As seen in the previous
section, single-molecule spectroscopy on freely-diffusing molecules has been able to
answer several questions in the dynamics of DNA-processing machines. Even so,
single-molecule spectroscopy using an immobilized format is necessary to watch an
entire process over an extended period. We were able to adapt previously successful
immobilization strategies to RNAP, to watch movement of RNAP with respect to the
DNA as a function of time [96]. The immobilization strategy was the same as that
used for Helicase experiments described by Professor Ha elsewhere in this topic [97].
The measurements on Helicase are an important example of measurements of
immobilized DNA-processing enzymes. We were also able to extend the ALEX
techniques to the TIR excitation/CCD imaging approach to single-molecule spec-
troscopy. In this way, themovement of RNAPwith respect to the DNAwasmonitored
during abortive initiation. The time scales could not be determined due to limitations
of the CCD frame rate. However, the system was clearly working on the surface
(Figure 9.12).
We would like to note that comparison of the results using immobilizedmolecules
with those from freely-diffusing molecules helped immensely in getting these
experiments to work.
9.4.2.3 Future Directions
There aremany possiblemethodological directions for this research; wemention two
examples. First, the interactions of additional proteins, or additional distances, may
be monitored with three-color methods [45
-
47]. Second, intracellular labeling
techniques may allow monitoring of transcription in vivo. If combined with the
high concentration methods outlined earlier, an extremely powerful methodology
