Chemistry Reference
In-Depth Information
70
before continuation of
transcription. There were however, steps in the puri
cation process that could have
taken off weakly bound
transcription was suggested that required release of
s
70
even if there were no obligatory release. Because of this,
there was an alternative model proposed that release of
s
70
was not necessary, but the
s
interactions were only weakened.
Recently, FRETmeasurements between the donor on
70
and acceptor on DNA
were used to determine whether there is obligatory release of
s
70
s
. Ensemble-level
experiments were performed
first [94], followed up by single-molecule measure-
ments using the same system [95]. This provides a good opportunity to compare
directly
the information content of
single-molecule and ensemble-level
measurements.
In both papers, a labeling scheme was used to monitor via FRET the movement of
RNAP. With trailing edge FRET, a donor label on
70
s
toward the back of the
70
complex undergoes FRET to an acceptor behind the RNAP (upstream
of the promoter). FRET ef
ciency starts high, and decreases as transcription
progresses. With leading edge FRET, a donor label on
RNAP
-
s
70
s
toward the front of the
70
complex undergoes FRET to an acceptor in front of the RNAP (down-
stream of the promoter). FRET ef
ciency starts low, and increases as transcription
progresses. In [94], ensemble FRET measurements were used to establish that a
signi
cant fraction of the
RNAP
-
s
70
remained attached to RNAP even to mature elongation
complexes. This contradicted some earlier models that maintained that the release of
s
s
70
was necessary to continue transcription. In this paper, the leading-edge FRET
measurements were critical, because they were able to differentiate between release
and non-release of
70
was still attached. However, the trailing-edge FRET measurements predicted a
decrease in FRET ef
ciency with or without release of
70
; if FRET increased with the progress of transcription, then
s
s
70
.
A primary advantage of single-molecule FRET measurements (with ALEX) over
ensemble FRETmethods is the ability to detect association of molecules even when
outside the range of FRET. This allows the detection of non-release of
s
70
even with
the trailing edge FRET measurements. We were able to determine the fraction of
complexes that retained
s
70
as a function of elongation.
This was an example of the power of single-molecule spectroscopy to determine
ordering of events, directly visualizing the heterogeneity of the system under study.
Even in this case, where ensemble measurements are able to answer some of the
basic questions, the single-molecule measurements provided signi
cant, quantita-
tive information that was otherwise unavailable. These tools have nowbeen applied to
a problem whose answer has remained elusive for over 25 years as we see next.
s
9.4.2.2 RNAP
-
Abortive Initiation
During the initiation of transcription, it takes several attempts to get started, to
prime the transcription pump. Abortive products (short RNA transcripts) are
formed in these initiation attempts, giving the name abortive initiation to the
process. Interestingly, DNA footprinting experiments indicating that during abortive
initiation, the RNAP was protecting the upstream boundary of the promoter (the
upstreamDNA element preceding the transcription start site, the binding site for the
