Biology Reference
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primer/template (p/t) binding to RT reveals two-exponential kinetics,
corresponding to a two-step mechanism (
Fig. 4.9
). The first phase
corresponds to the formation of a RT-p/t collisional complex inducing
a conformational transition of RT from the closed- to the open-
polymerase typical right-hand organization. The on rate (
k
þ
1
) of the first
phase is dependent on the concentration of RT and is extrapolated from
the slope of the secondary plot of
k
obs1
versus enzyme concentration.
The off rate (
k
1
) can be estimated from the same plot from the
intercept on the
k
obs
axis. This first step is followed by conformational
changes of the preformed RT-p/t complex, which correctly places the
nucleic acid in the appropriate binding site for catalysis. The
k
2
(
k
þ
2
k
2
) rate constant of the second phase is independent of the
enzyme concentration and corresponds to the second exponential term of
the kinetics.
40,41,45
By combining FRET and single-molecule fluorescence, Patel and col-
leagues have elucidated the initiation of transcription catalyzed by RNA
polymerase
46,47
using two double-stranded DNA strands corresponding to
the upstream promoters and downstream template DNA, labeled at different
positions with two different dyes. The donor dye is associated to the
upstream promoter and the acceptor to the template strand. Essential
changes in DNA and polymerase conformation associated with initiation
and abortive RNA synthesis have been identified, and a mechanism
involving DNA scrunching following by its rotation has been proposed for
early initiation catalyzed by T7 RNA polymerase. Similarly, combining
single- and double-stranded labeled DNA offers a powerful sensing system
to monitor the open/closed conformational transition of the RNA or DNA
polymerase during initiation as well as nucleotide incorporation in real time
and the walking of the polymerase along the DNA or RNA during
elongation, as reported for mitochondria RNA polymerase
48
and for HVC
polymerase (NS5B).
49
þ
2.3. Peptide/nucleic acids
During the last two decades, understanding peptide/nucleic acids interac-
tions has become a major challenge in the field of drug delivery. Small
peptides called cell-penetrating peptides (CPPs) have been developed to
improve the cellular internalization of a wide range of nucleic acids (from
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