From the Department of Physics and
§ Department of Molecular and Cell Biology, University of
California, Berkeley, California 94720 and the
Computer Science Department and Department of
Physics and Astronomy, University of North Carolina,
Chapel Hill, North Carolina 27599
In Escherichia coli, transcription is
carried out by a single enzyme, E. coli RNA polymerase
(RNAP),1 and the rate of
transcription initiation is controlled, in part, by the rate at which
the polymerase can find the promoter. One problem that RNAP, and for
that matter most other specific DNA-binding proteins, must solve is how
to overcome the kinetic barrier of finding its specific binding site
amid a large excess of nonspecific DNA. In 1968, Adam and
Delbrück (1) proposed that the efficiency of a
diffusion-controlled search could be enhanced by orders of magnitude if
it were to take place in a space of lower dimensionality. In 1970, Riggs et al. (2) reported that E. coli lac
repressor locates its target site at rates up to 1000 times faster than what could be accounted for by a three-dimensional diffusion-controlled search. These authors suggested that, because the enzyme has some affinity for non-promoter DNA, their observations could be explained if
the enzyme could bind nonspecifically anywhere to the DNA and then move
in a one-dimensional random walk along the DNA until it finds its
target. This finding sparked the interest of scientists to demonstrate
the existence and to elucidate the mechanisms of facilitated target
location in DNA-binding proteins.
Two investigations have used kinetic analysis to obtain evidence that
RNAP can locate a promoter by one-dimensional diffusion along
nonspecific DNA. Singer and Wu (3) employed a rapid
mixing/photocross-linking method to monitor the
time-dependent density of bound RNAP along a relaxed,
circular DNA plasmid containing a single promoter. It was found that
the occupancy by RNAP of the DNA segments near a promoter decreased
faster than the occupancy of the segments farther away from the
promoter. This phenomenon was interpreted as evidence that RNAP reached
the promoter through one-dimensional diffusion along the DNA. Fitting
the data to a theoretical model that included RNAP sliding, a
dissociation rate constant of koff = 0.3 s In two recent studies, fluorescence microscopy was used to observe the
interactions of fluorescently labeled RNAP with DNA. Kabata et
al. (6) utilized superintensified fluorescence microscopy to
visualize the movement of RNAP over Other mechanisms of facilitated target location besides
sliding have been proposed, as depicted in Fig.
1. One mechanism involves the transfer of
DNA-binding proteins from one segment of DNA to another. This process,
known as intersegment transfer (8), could make it more
likely for a protein molecule to bind to distant regions in the genome
by decreasing the effective volume of diffusion and thus increasing the
target location rate. Moreover, as pointed out by von Hippel and Berg
(8), the efficiency of this process can be enhanced if the energy
barrier of intersegment transfer is lower than the barrier for the
dissociation-association cycle. In particular, the high local
concentration of DNA would also make contact increasingly likely and
multiple transfer events possible.
INTRODUCTION
TOP
INTRODUCTION
Scanning Force Microscopy...
Conclusions and Summary
REFERENCES
1 and a one-dimensional diffusion coefficient of
D1D = 1.5 × 10
9
cm2/s for RNAP sliding along DNA were determined. The
resulting average lifetime of a nonspecific complex,
av = 3.3 s, is, however, about 1000 times larger than the one found
in electron microscopy experiments (4). In the second study, Ricchetti
et al. (5) measured the occupancy of DNA fragments carrying
A1 promoters as a function of the length of the downstream and upstream
flanking sequences. It was found that a longer downstream flanking
sequence increased the occupancy of the adjacent promoter in agreement
with the sliding model. However, upstream sequences had surprisingly
little effect on promoter occupancy.
-DNA combs. A fraction of the
RNAP molecules was seen to deviate from the direction of bulk flow and
to move along the extended DNA molecules. This observation suggests
that RNAP can slide along nonspecific DNA. However, in this experiment
the RNAP was propelled predominantly by flow and was, therefore, not
driven by thermal motion. In the second study, Harada et al.
(7) used internal reflection fluorescence microscopy to observe the
dissociation and association events of RNAP with different regions of a
single
-DNA molecule, which was suspended in laser tweezers. For
AT-rich regions fast and slow dissociation constants of 3.0 and 0.66 s
1, respectively, were determined; and for GC-rich
regions a fast dissociation rate of 8.4 s
1 was measured.
In a few instances sliding of RNAP along the DNA was also observed.
However, the occurrence of these events was rare because the present
spatial resolution of this technique is about 200 nm, and the typical
sliding range of a one-dimensionally diffusing RNAP is presumably less
than this limit. Nevertheless, a one-dimensional diffusion constant of
~10
10 cm2/s was estimated from these data.
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Fig. 1.
Schematic of the proposed mechanisms of
facilitated target location, where the ovals represent RNA
polymerase molecules and the curved line represents the
DNA. a, sliding is the one-dimensional
diffusion of the RNAP along the contour of the DNA. b,
intersegment transfer is the translocation of the RNAP from
one point of the DNA to another distant point through the formation of
a ternary structure. c, intradomain association and
dissociation or "hopping" is the movements of the
RNAP relative to the DNA through a series of collisions on a small
scale.
A third proposed mechanism is the intradomain association and
dissociation or "hopping," of the
protein along the DNA (8). In this process, the protein-DNA
interactions may consist of the protein bouncing along the DNA until it
finds the target site or completely dissociates from the DNA. The
target search would then be accelerated in a manner similar to sliding,
with a reduced number of sampled sequences but presumably higher
kinetic barriers.
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Scanning Force Microscopy Studies |
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This review will center on the capability of the scanning force microscope (SFM) to operate in liquids to directly visualize the movements of E. coli RNA polymerase on nonspecific DNA, thus providing direct evidence for the mechanisms of facilitated target location. By necessity, this review will focus on experiments performed mainly in our laboratory, but the results will be compared with those obtained by other techniques. In experiments of this kind, the DNA molecules are deposited on a mica surface, where they interact with other molecules (i.e. RNAP) present in the solution covering the surface. To study these interactions properly it is necessary to understand the state of the DNA on such surfaces and to control the mobility of the DNA molecules.
DNA Diffusion on Mica Surface-- Imaging the dynamic interactions between RNAP and DNA by SFM requires first finding conditions in which the protein-DNA complexes are adsorbed stably enough to be imaged by the scanning tip. Yet, these same conditions must allow the molecules to bind loosely enough to be able to diffuse on the substrate. Deposition conditions had to be found to reconcile these conflicting requirements. As reported previously (9-11), binding of DNA to the mica surface requires the presence of a divalent cation such as Mg2+, Mn2+, Ca2+, Zn2+, or Ni2+ in the deposition buffer. Magnesium was chosen as the divalent ion in the experiments to be described herein, as it is also essential for RNAP activity, whereas other cations are inhibitory to transcription (Zn2+, Ni2+) (12) or induce kinks in the DNA (Zn2+) (13). Images were acquired under liquid using tapping mode SFM. Electron beam-deposited tips (14) were used in all experiments. These deposition and imaging conditions favor the binding of the DNA molecules to the mica, while allowing them to diffuse laterally.
In Fig. 2, the contours of free DNA
molecules (1047 bp) from 11 sequentially recorded images are
superimposed to illustrate the DNA motion on the mica surface. The
excursion of the center of mass of one of the DNA molecules is shown in
the inset of Fig. 2. The average two-dimensional diffusion
coefficient, determined from 91 molecules observed in several different
experiments, had a value of about 7 × 1014
cm2/s. The diffusion coefficient varied by approximately 1 order of magnitude among different experiments. This is probably
because of the local variability of the mica surface, which influences the strength of the mica-DNA interactions and thus the mobility of
DNA.
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Diffusion of RNA Polymerase along Nonspecific DNA--
Having
obtained conditions in which DNA molecules can diffuse on the substrate
surface while remaining adsorbed to it, experiments were designed to
investigate whether RNAP can diffuse one-dimensionally along
nonspecific DNA. The remainder of this article will be devoted to
describing those experiments and analyzing the results in comparison with the limited amount of information available in the literature. Complexes of RNAP holoenzyme formed on a promoterless DNA fragment were
deposited onto mica and imaged in buffer with the SFM. Fig. 3 shows a time-lapse sequence of a
nonspecific RNAP-DNA complex. At the center of each image the
polymerase appears as a white globular feature stably bound to the
surface. The DNA molecule appears to be sliding back and forth beneath
the enzyme (Fig. 3, A-G). In the last frame (Fig.
3H), the protein has released the DNA. The position of the
RNAP on the DNA fragment in successive images is depicted schematically
in Fig. 3I. Similar plots, obtained from over 30 different
imaging sequences, revealed that the relative position of the protein
on the DNA is consistent with the pattern of a random walk in one
dimension. The average one-dimensional diffusion coefficient obtained
from these images has a value of D1D = 1.1 × 1013 cm2/s. The average lifetime of these
nonspecific RNAP-DNA complexes on the surface was about 600 s,
which is large compared with nonspecific complexes in solution (see
below).
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Effect of Heparin on Nonspecific Complexes-- To demonstrate that the observed RNAP-DNA complexes are indeed nonspecific complexes, and not just the DNA and RNAP positioned in close proximity by chance, a control experiment was carried out by introducing heparin into the liquid chamber. Heparin, a polyanion and DNA analog, is known to disrupt the relatively weak nonspecific RNAP-DNA interaction and to inhibit DNA binding of RNAP (15-17). Accordingly, if these complexes were true nonspecific complexes, heparin should have a deleterious effect on sliding. On the other hand, heparin would have no effect if RNAP and DNA were not interacting with each other or if the complexes were the heparin-resistant tight binding kind (15). When 14 sliding, nonspecific complexes were exposed to heparin, 13 of them dissociated right after the arrival of heparin in the liquid chamber, and no re-association was observed; only one of them seemed to stay bound (data not shown). These results showed that heparin significantly reduced the average lifetime of the observed DNA-RNAP complexes, providing additional proof that they were indeed nonspecific complexes.
Intersegment Transfer--
Fig. 4
shows a series of images where RNAP core enzyme combines sliding with
an intersegment transfer event. Intersegment transfer is not
nearly as common as sliding, probably because of the low concentration
of DNA on the surface. The images show an intermediate structure
consisting of an RNA polymerase molecule binding two segments of the
same DNA molecule (Fig. 4C). An interesting feature of the
intersegment transfer event is the formation of a tight
hairpin turn in conjunction with the formation of the ternary
complex.
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Intradomain Association and Dissociation (Hopping)--
The images
in Fig. 5 show that an RNA polymerase
molecule, initially bound to the DNA, dissociated from the DNA and then
re-associated. The series of events depicted by these images indicate
that, in the process of searching for the promoter, the polymerase
might associate to and dissociate from the DNA repeatedly until the target site is found. At present, the temporal resolution of the SFM
does not allow for a more detailed observation of these events. The
rates of association and dissociation of the RNA polymerase in solution
are much higher than the time resolution of the images, and it is
possible that between images multiple association-dissociation cycles
occur.
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Conclusions and Summary |
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In the present study, sequential SFM images of single RNAP molecules moving (sliding, intersegment transfer, and possibly hopping) along individual DNA molecules were recorded and analyzed. These images demonstrate that RNAP can use one-dimensional diffusion along nonspecific DNA, as well as intersegment transfer and hopping, to accelerate the search for promoters. The ability of heparin to disrupt the sliding process and, thereby, reduce the mean diffusion time of the polymerase on the DNA supports the identification of the RNAP-DNA complexes as nonspecific complexes and eliminates the possibility that these complexes are simply DNA and RNAP juxtaposed by chance. Furthermore, this observation indicates that the sliding, nonspecific complexes are not the tight binding type previously described for polymerase (15) because those complexes cannot be dissociated by heparin.
The average lifetime of surface-bound nonspecific complexes was found
to be about 600 s, which is much larger than the average lifetime
of 3.3 s (3) and 1.5-0.1 s (7) reported for nonspecific complexes
in solution under similar salt conditions. A possible explanation is
that the constraints imposed by the surface slow down the rates at
which the complex adopts intermediate configurations required for
dissociation. Dissociation rates smaller by a factor of 180 would
require an increase in the activation energy leading to this
intermediate by about 3.1 kcal/mol. This value is well within the range
of activation energies for surface diffusion of DNA (~8.1 kcal/mol)
obtained from a comparison between the diffusion constants of the DNA
fragment on the surface, D2D = 7·1014 cm2/s, and in solution,
D3D = 5.4·10
8 cm2/s
(18). Furthermore, both molecules remain in close proximity to each
other for a longer time when RNAP releases the DNA, because both of
them are adsorbed to the surface. Attractive interactions between the
molecules might increase the probability that RNAP and DNA re-associate
many times before the DNA fragment eventually dissociates completely
and diffuses away. This interpretation is consistent with the
observation that the lifetime of nonspecific complexes is reduced in
the presence of the negatively charged DNA competitor, heparin. It is
possible that the small diffusion constants and large lifetimes
observed on the surface might also be observed in analogous experiments
that were carried out in very viscous medium such as the inside of
cells (19).
In summary, images obtained with tapping-mode SFM could demonstrate the
diffusion of E. coli RNA polymerase along DNA. Moreover, direct evidence of other mechanisms of facilitated targeting of RNAP
such as intersegment transfer and possibly
hopping (intradomain association and
dissociation) was obtained for the first time. Ternary
intermediates, in which RNAP appears to be simultaneously bound to two
DNA segments, were directly observed during intersegment transfer events. In addition, these transfer events were preceded and followed by sliding processes in some experiments. A combination of
intersegment transfer, hopping, and sliding
should result in a more effective search of the promoter. Even though
it is not possible to directly compare numerical data from the SFM
study with kinetic data from solution studies, the current analysis provides the first direct evidence on individual complexes, at high
resolution, for these targeting mechanisms. Future developments in SFM
technology should allow faster scanning of biological samples and
thereby improve the temporal resolution of these SFM movies. These
improved capabilities, together with single-molecule analysis in real
time, will make it easier to use SFM to visualize complex biological processes as they occur, one molecule at a time.
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FOOTNOTES |
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* This minireview will be reprinted in the 1999 Minireview Compendium, which will be available in December, 1999. This the the fourth article of four in the "Biochemistry at the Single-molecule Level Minireview Series."
¶ To whom correspondence should be addressed. Tel.: 510-643-9706; Fax: 510-642-5943; E-mail: carlos{at}alice.berkeley.edu.
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ABBREVIATIONS |
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The abbreviations used are: RNAP, RNA polymerase; SFM, scanning force microscope; bp, base pairs.
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REFERENCES |
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