368
Biochemical Society Transactions (2013) Volume 41, part 1
Modern biophysical approaches probe
transcription-factor-induced DNA bending and
looping
Andreas Gietl and Dina Grohmann1
Physikalische und Theoretische Chemie–NanoBioSciences, Technische Universitát Braunschweig, Hans-Sommer-Strasse 10, 38106 Braunschweig, Germany
Abstract
The genetic information of every living organism is stored in its genomic DNA that is perceived as a chemically
stable and robust macromolecule. But at the same time, to fulfil its functions properly, it also needs to be
highly dynamic and flexible. This includes partial melting of the double helix or compaction and bending of
the DNA often brought about by protein factors that are able to interact with DNA stretches in a specific and
non-specific manner. The conformational changes in the DNA need to be understood in order to describe
biological systems in detail. As these events play out on the nanometre scale, new biophysical approaches
have been employed to monitor conformational changes in this regime at the single-molecule level. Focusing
on transcription factor action on promoter DNA, we discuss how current biophysical techniques are able to
quantitatively describe this molecular process.
The shaped DNA landscape
In 1953, Watson and Crick presented the three-dimensional
structure of DNA revealing its double-stranded configuration and its physicochemical properties [1]. The discovery
that DNA is compacted into nucleosomes that form higherorder structures led to the current view that DNA is not a
stiff but a flexible polymer [2–4]. Bending, wrapping and
looping of DNA are reoccurring themes in life and are
not restricted to the compaction of DNA by histones or
histone-like proteins, but are also common to transcriptional
regulation in all domains of life. TFs (transcription factors)
can cause sharp bends and kinks in the DNA [5–7], and
transcriptional enhancers very often cause looping of the
DNA to allow long-range interactions between genetic
elements [8]. Looking at the overall structural changes in
the DNA, the protein-induced remodelling of the DNA
curvature seems to be severe. But quantification of these
changes and an analysis of the dynamic properties of the
nucleoprotein complexes are challenging as they happen at
the sub-nanometre regime. However, information obtained
using single-molecule techniques have complemented our
understanding of protein-induced conformational changes in
DNA as these techniques provide sub-nanometre sensitivity
often with a high temporal resolution. In the present paper,
we provide a short insight into modern biophysical methods
Key words: atomic force spectroscopy (AFM), molecular tweezers, single-molecule fluorescence
spectroscopy, TATA-box-binding protein (TBP), tethered particle motion (TPM), transcription
factor.
Abbreviations used: AFM, atomic force microscopy; EM, electron microscopy; FRET,
Förster/fluorescence resonance energy transfer; HMGA1, high-mobility group A1; NC2, negative
cofactor-2; PIC, preinitiation complex; RNAP, RNA polymerase; smFRET, single-molecule FRET;
TBP, TATA-box-binding protein; TF, transcription factor; TPM, tethered particle motion.
1
To whom correspondence should be addressed (email [email protected]).
C The
C 2013 Biochemical Society
Authors Journal compilation that are helpful tools to gain information on the structure,
kinetics and thermodynamics of DNA elasticity. In order
to illustrate the depth and variety of information that can
be gained by these often complementary methods, we chose
the interaction of the general archaeal-eukaryotic TF TBP
(TATA-box-binding protein) with the promoter DNA as an
example.
TBP: a conserved general TF
Multisubunit RNAPs (RNA polymerases) are not capable
of starting transcription by themselves, but rely on basal
TFs that direct the RNAP to the promoter DNA upstream
of the transcription start site. The general TFs TBP and
TFB are necessary and sufficient to start RNAP-directed
transcription and are highly conserved in structure and
function in archaea and eukaryotes. TBP specifically binds to
a A-T-rich sequence named the TATA-box (DNATATA ) which
is found in the core promoter of protein-encoding genes
[9,10]. TFB recognizes the DNA–TBP complex, stabilizes
the TBP–DNA interaction [11–13] and is able to form a
ternary complex composed of TBP, TFB and DNATATA .
TFB furthermore recruits the RNAP to the promoter
eventually forming the PIC (preinitiation complex) [14,15]
ensuring the site-specific start of transcription. Even though
eukaryotic TBP is part of the multisubunit TFIID, it can
act independently of the 13–14 proteins that constitute
TFIID. Eukaryotic TBP additionally contains an N-terminal
domain not present in archaeal TBP, hinting at the possibility
that the eukaryotic transcription machinery can accomplish
transcriptional regulation via the N-terminal part of TBP [16].
Both archaeal and eukaryotic TBP bind DNATATA with high
affinity [17–19]. Archaeal TBP and the C-terminal part of
Biochem. Soc. Trans. (2013) 41, 368–373; doi:10.1042/BST20120301
Molecular Biology of Archaea 3
the eukaryotic TBP exhibit a highly conserved saddle-like
structure with two-fold symmetry most likely to be the result
of a gene-duplication event [20–22] (Figure 1A). Interestingly,
TBP does not undergo a significant restructuring upon
interaction with the DNA and TFB. In contrast, binding of
TBP induces a sharp bend in the DNATATA and structural
studies carried out on the archaeal or eukaryotic machinery
revealed that two conserved phenylalanine pairs are inserted
between the first and last two bp of the TATA-box [23–25].
The insertion into the minor groove of the DNA leads to
a widening of the double-stranded helix and results in two
sharp kinks towards the major groove at the two ends of the
TATA-box (Figure 1A).
FRET (Förster/fluorescence resonance energy transfer)
has been used extensively to monitor the formation of the
DNA–TBP complex. FRET occurs between a donor and an
acceptor fluorophore and the main factors that determine the
efficiency of the energy transfer are (i) the degree of spectral
overlap between the emission and excitation spectrum of the
donor and acceptor, (ii) the quantum yield of the donor,
and (iii) the distance between donor and acceptor. Because
of its strong distance-dependency, FRET is often referred
to as a ‘spectroscopic ruler’ that allows the measurement of
inter- and intra-molecular distances in the range 2–8 nm [26–
28]. The bending of the DNA upon TBP association can be
easily monitored using FRET when a donor and acceptor
fluorophore are attached to the DNA at either side of the
TATA-box, as the two DNA arms come into close proximity
upon bending, resulting in an increased energy transfer. The
Parkhurst laboratory employed this FRET assay extensively
to monitor the association and dissociation kinetics of the
DNA–TBP complex using mainly time-resolved stoppedflow measurements [29]. They showed that DNA binding
and bending by TBP is rapid and occurs simultaneously
[29]. Further studies led to a three-step linear kinetic model
for the human and yeast TBP–DNATATA interaction as
shown in Figure 1(B) [30,31]. A small population of prebent DNA binds to TBP, with two intermediate conformers
(I1 and I2 ) apparent along the pathway to formation of
the final TBP–DNA complex [19]. On the basis of the
distance measurements via FRET, a bending angle of 82◦
for yeast TBP and 97◦ for human TBP could be determined
using the adenovirus major late promoter. This is in
excellent agreement with the 80◦ bend determined in highresolution crystallographic studies. Notably, the degree of
DNA bending for the intermediate states has been found to
be the same as for the final state. But, unlike that seen in the Xray structures, the studies carried out in solution revealed that
the bending angle largely depends on the TATA sequence and
the TBP species used ranging from 32◦ to 97◦ and correlates
with the transcription efficiency of the promoters used [31].
The authors hypothesize that the bend angle defines
the positioning of subsequent TFs. FRET has also been the
method of choice to demonstrate that the eukaryotic TFIIA
alters the conformation of TBP bound to DNATATA and
increases the kinetic stability of the TBP–DNATATA complex
[32].
Visualizing DNA topology using AFM
(atomic force microscopy)
Taking a picture of individual altered DNA strands would be
the most direct approach to ‘see’ DNA bending and looping.
But as the dimensions of DNA do not exceed the nanometre
range, specialized microscopy techniques such as EM (electron microscopy) must be employed to reach the resolving
power needed. Sample preparation is quite elaborate in
EM studies and often involves harsh fixation or freezedrying treatments. Over the last 20 years, AFM has reached
a sensitivity that allows the three-dimensional exploration of
biological samples. Using AFM, it is possible to quantify
the force that is applied to a tiny tip on a hook spring
(cantilever) by monitoring the deflection of a laser beam
from the top of the tip [33]. Three different scan types are
commonly used: constant cantilever height, constant applied
force and the tapping mode. The tapping mode is usually
used for soft matter investigations (e.g. proteins and DNA)
in liquid buffers because the tip is not required to touch the
fragile sample. A piezo applies a high-frequency oscillation
in the z-direction to the cantilever. When scanning a sample
which is immobilized on a mica surface, the distance between
the surface and the tip decreases and, at the same time,
attracting forces (e.g. van der Waals force) increase and result
in amplitude decrease of the oscillation. This readout is highly
sensitive and even nanometre-sized structures such as the
DNA helix can be resolved under physiological conditions
[34] (Figure 2A). In contrast with EM, sample preparation for
AFM is fast and allows buffers near physiological conditions.
AFM has been widely applied to investigate the flexibility of
the DNA (e.g. [35–37]) even allowing time-lapse studies that
monitored the movement of T7 and bacterial RNAP on its
DNA substrate [38–40].
AFM also enabled the visualization of the complex eukaryotic IL2RA (interleukin 2 receptor α) gene
promoter DNA [41,42], which contains a functional
and non-functional TATA-box as well as two regulatory regions with binding sites for nucleosomes and
TFs. The images of the ‘naked’ DNA showed a curved
and flexible structure at the site of the functional TATA-box,
whereas the non-functional sequence appeared to be straight
and rigid, supporting the idea that the pre-bent state of the
DNATATA enables the recognition by TBP [43]. Furthermore,
time-lapse studies revealed that the addition of the nonhistone chromatin protein HMGA1 (high-mobility group
A1) induces a remodelling of the DNA structure caused
by the association of HMGA1 with the supercoiled part of
the promoter DNA, leading to the ejection of a nucleosome
positioned at the promoter. On the basis of these results,
the authors suggest that, in the presence of HMGA1, the
promoter is activated as the binding of the general TFs TBP
and TFIIB is enabled after the ejection of the nucleosome.
Complementary single-molecule methods also based on
force measurements are optical and magnetic tweezers
(Figure 2D). Here, the sample is either tethered to the surface
and a bead or the sample is spanned between two beads.
C The
C 2013 Biochemical Society
Authors Journal compilation 369
370
Biochemical Society Transactions (2013) Volume 41, part 1
Figure 1 Structure and kinetic model of the TBP–DNA interaction
(A) Structure of the archaeal TBP–TFB–DNA complex [TBP (PDB code 1AIS), orange; TFB, red; TATA-box, green; DNA, grey;
conserved phenylalanine residues, blue]. The TFB core domain (red) stabilizes the TBP–DNA complex, but does not influence
the bending angle. (B) Proposed model of the interaction between eukaryotic TBP and promoter DNA based on kinetic data
(colouring as in A). TBP binds the promoter DNA recognizing the TATA-box motif. Insertion of four conserved phenylalanine
residues into the DNA results in a severe conformational distortion of the DNA. The kinetic data suggest a three-step binding
scenario and predict two intermediate states along the pathway to a final complex that might differ in the phenylalanine
insertion pattern. The intermediate and final states have been postulated to vary in their stability, but do not show a different
bending angle.
A pulling force is generated on the bead by an optical
or magnetic field gradient in order to keep the bead at a
constant height above the pre-stretched DNA. Following the
bead’s diffraction rings (their count and diameter depends
on the bead’s z-position), a movement can be detected with
nanometre sensitivity. Tweezers allow the measurement of
forces as low as piconewton-scale generated by, e.g., proteins
that act on DNA molecules. Upon conformational changes
in the polymer, the location of the bead changes and the force
generated can be quantified with a temporal resolution in
the low-millisecond range [44]. This method has not been
applied to the TBP–DNA interaction yet, but could inform
about the forces generated by TBP when bending the DNA
and could quantify the need for a pre-bent DNA for TBP
recognition. Furthermore, hypothetical scenarios could be
tested revealing, for example, whether the strained DNA
conformation supports TBP dissociation [45].
Assessing DNA flexibility changes using
TPM (tethered particle motion)
TPM can be viewed as a tracking method. In TPM, a bead
of (sub-)micrometre size is attached to one end of a polymer
strand that has been attached to a glass surface at the opposite
end of the polymer. Both bead and polymer are in solution
and, consequently, the motion of the bead is governed
by Brownian motion of the polymer. The movement of
the bead is recorded using CCD (charge-coupled device)
C The
C 2013 Biochemical Society
Authors Journal compilation based video microscopy, and allows the calculation of spring
constants and other mechanical properties of the tethering
biopolymer (Figure 2B). This technique can be applied to
DNA–protein interactions as the motion of the bead is
sensitive to changes in the polymer length (DNA being
the polymer) that are induced by, e.g., DNA bending [46].
Tolić-Nørrelykke et al. [47] tethered a polystyrene bead to
TATA-box-containing DNA and measured the amplitude of
Brownian motion in the presence and absence of yeast TBP
[47]. The binding of TBP led to a reduction in the Brownian
motion compared with that of unbent DNA. Using TPM, a
low-spatial, but high-temporal, resolution can be achieved so
that the TBP-binding and -unbinding steps could be clearly
distinguished, yielding dissociation and association rates for
the individual events. In addition to the well-defined free
and bound classes of Brownian motion, two more classes of
motion representing two additional conformers of the TBP–
DNA complex could be observed. The kinetic parameters of
these extra classes match the ensemble fluorescence data that
identified the two intermediate states. Therefore the singlemolecule experiments support the three-step linear binding
mechanism.
Following bending dynamics using
single-molecule fluorescence spectroscopy
As equally interesting as the topological changes in the
DNA are the dynamics of the TBP–DNATATA interaction,
Molecular Biology of Archaea 3
Figure 2 Single-molecule techniques image and probe the TBP–promoter DNA interaction
(A) Simplified model of AFM showing its core components: cantilever, laser beam and a quadrant diode that detects the
status of the cantilever via the deflection of a laser beam. The AFM cantilever tip scans a mica surface and the immobilized
sample line by line. Inset: the free promoter DNA is either not or only weakly bent, whereas TBP binding leads to significant
kinks (orange zone). (B) Sketch of a TPM set-up. The surface–bead distance is shortened once TBP (orange) induces DNA
bending, which in turn leads to a reduced amplitude of the bead’s random walk. The motion of the micrometre bead is
tracked with a camera and the bead position is plotted over time (lower left). The black area denotes the particle’s motion
with the extended DNA, whereas the inner orange area can be assigned to limited motion caused by bending the DNA by
TBP. Plotting the Brownian motion [root mean square deviation (RMSD)] against time allows the identification of TBP-binding
events (orange areas in the graph). (C) SmFRET experiments require the labelling of the promoter DNA with a donor and an
acceptor fluorophore adjacent to the TATA-box. DNA bending by TBP shortens the distance between the two fluorophores,
leading to an increase in FRET, i.e. the donor intensity (green) decreases while the acceptor intensity (red) increases (upper
trace). The lifetime of the TBP–DNA complex (orange areas) can be derived from the resulting FRET efficiency plot. (D) A
magnetic tweezers set-up. A super-paramagnetic bead is pulled by a magnetic field to stretch the DNA. The bead is pulled
towards the surface when TBP bends the DNA. The bending events are shown in the transient below (orange box). All
transients shown in (A)–(D) are theoretical simulations according to the data presented in the literature.
which can be accessed using smFRET (single-molecule
FRET) measurements. Monitoring FRET efficiency and
fluorescence intensity of individual molecules provides
valuable information about heterogeneous populations as
(i) biologically relevant subpopulations can be identified
according to FRET efficiency and label stoichiometry, (ii)
synchronization of the molecules is not necessary to study the
molecular kinetics or the stochastic behaviour of biological
C The
C 2013 Biochemical Society
Authors Journal compilation 371
372
Biochemical Society Transactions (2013) Volume 41, part 1
processes, and (iii) real-time imaging of single molecules
at high-temporal resolution (millisecond range) is possible
(Figure 2C). SmFRET measurements have been carried out
using the TBP–DNA complex. Here, the donor and acceptor
emission fluorescence of immobilized DNATATA molecules
was recorded simultaneously using TIRF (total internal
reflection fluorescence) microscopy and the FRET efficiency
could be calculated from the fluorescence intensity traces
[48–50]. In the study conducted by Blair et al. [50], human
TBP causes a homogeneous bent state and does not lead to
partially bent DNA. In contrast with the ensemble studies,
the bending angle was not influenced by the DNA sequence
and not affected by TFIIB. However, TFIIB is able to shift
the equilibrium towards the TBP–DNA complex.
A more detailed analysis of the smFRET measurement
allows the calculation of association and dissociation kinetics
as well as complex lifetimes. Comparing the dynamics of the
archaeal TBP–DNATATA interaction with the data collected
from the human and yeast system, it becomes clear that
there is a notable difference in complex lifetimes. Using TBP
from the hyperthermophilic organism Methanocaldococcus
jannaschii we showed that the TBP–DNATATA interaction
is highly dynamic with fast binding and unbinding events
[49]. The TBP–DNA complex dissociates rapidly within
milliseconds, whereas the human and yeast TBP forms verylong-lived complexes with dwell times up to several minutes
[48,50].
Making use of a labelled TBP variant, further insights
could be gained with respect to the movement of TBP along
the DNA. Monitoring a FRET signal between an acceptor
fluorophore attached to the DNA and a donor positioned
in TBP, it was demonstrated that NC2 (negative cofactor2) induces dynamic conformational changes in the TBP–
DNA complex indicated by two distinct FRET populations.
Changing the conformation of TBP–DNA complexes allows
TBP to move along the DNA and to escape from the TATAbox. In this model, the association of TFIIB at the TATA-box
is inhibited and therefore leads to enhanced transcriptional
repression by NC2 [48].
Summary and outlook
Biophysical studies revealed that the TBP–DNATATA interaction is driven by structural and dynamic aspects. The intrinsic
conformation of the promoter DNA influences the formation
of the TBP–DNATATA complex as much as additional TFs
do, which can lead to either activation or repression of
transcription. General TFs such as TFIIA and TFIIB/TFB
shift the equilibrium towards the final TBP–DNA complex,
resulting in an increased transcription initiation rate. In
contrast, specific TFs can work both ways. PIC formation
is enhanced once nucleosomes and chromatin-like proteins
are repositioned by these additional TFs. On the other hand,
factors such as NC2 are able to repress transcription by
mobilizing TBP and preventing the formation of stable PICs.
As single-molecule measurements provide a high temporal
resolution, the lifetime of TBP–DNATATA complexes could
C The
C 2013 Biochemical Society
Authors Journal compilation be determined recently. The lifetime of the eukaryotic
(minutes to hours) and archaeal (milliseconds) complexes
differ greatly, and further studies are needed in order to
understand how complex lifetime influences transcriptional
activity in these two domains of life.
Acknowledgements
We thank A. Gust, P. Holzmeister, M. Overhoff, E. Pibiri, S. Schulz and
A. Zander for helpful discussions and a critical reading of the paper.
We are grateful to P. Tinnefeld for his support. We apologize to
colleagues whose work could not be cited owing to space limitations.
Funding
Research in D.G.’s laboratory was supported by the German Research
Foundation [grant number GR 3840/2-1] and by the German–
Israeli Foundation Young Scientist programme [grant number 22922264.13/2011].
References
1 Watson, J.D. and Crick, F.H. (1953) Molecular structure of nucleic acids; a
structure for deoxyribose nucleic acid. Nature 171, 737–738
2 Olins, A.L. and Olins, D.E. (1974) Spheroid chromatin units (v bodies).
Science 183, 330–332
3 Thomas, J.O. and Kornberg, R.D. (1975) An octamer of histones in
chromatin and free in solution. Proc. Natl. Acad. Sci. U.S.A. 72,
2626–2630
4 Woodcock, D.M. and Sibatani, A. (1975) Differential variations in the
DNA of Drosophila melanogaster during development. Chromosoma 50,
147–183
5 Peeters, E., Willaert, R., Maes, D. and Charlier, D. (2006) Ss-LrpB from
Sulfolobus solfataricus condenses about 100 base pairs of its own
operator DNA into globular nucleoprotein complexes. J. Biol. Chem. 281,
11721–11728
6 Kapanidis, A.N., Ebright, Y.W., Ludescher, R.D., Chan, S. and Ebright, R.H.
(2001) Mean DNA bend angle and distribution of DNA bend angles in
the CAP–DNA complex in solution. J. Mol. Biol. 312, 453–468
7 Wong, O.K., Guthold, M., Erie, D.A. and Gelles, J. (2008) Interconvertible
lac repressor–DNA loops revealed by single-molecule experiments. PLoS
Biol. 6, e232
8 Sanyal, A., Lajoie, B.R., Jain, G. and Dekker, J. (2012) The long-range
interaction landscape of gene promoters. Nature 489, 109–113
9 Zhang, J., Li, E. and Olsen, G.J. (2009) Protein-coding gene promoters in
Methanocaldococcus (Methanococcus) jannaschii. Nucleic Acids Res. 37,
3588–3601
10 Breathnach, R. and Chambon, P. (1981) Organization and expression of
eucaryotic split genes coding for proteins. Annu. Rev. Biochem. 50,
349–383
11 Werner, F. and Weinzierl, R.O. (2005) Direct modulation of RNA
polymerase core functions by basal transcription factors. Mol. Cell. Biol.
25, 8344–8355
12 Hausner, W., Wettach, J., Hethke, C. and Thomm, M. (1996) Two
transcription factors related with the eucaryal transcription factors
TATA-binding protein and transcription factor IIB direct promoter
recognition by an archaeal RNA polymerase. J. Biol. Chem. 271,
30144–30148
13 Bell, S.D. and Jackson, S.P. (1998) Transcription and translation in
archaea: a mosaic of eukaryal and bacterial features. Trends Microbiol. 6,
222–228
14 Grohmann, D. and Werner, F. (2011) Recent advances in the
understanding of archaeal transcription. Curr. Opin. Microbiol. 14,
328–334
15 Hahn, S. and Young, E.T. (2011) Transcriptional regulation in
Saccharomyces cerevisiae: transcription factor regulation and function,
mechanisms of initiation, and roles of activators and coactivators.
Genetics 189, 705–736
Molecular Biology of Archaea 3
16 Adachi, N., Natsume, R., Senda, M., Muto, S., Senda, T. and Horikoshi, M.
(2004) Purification, crystallization and preliminary X-ray analysis of
Methanococcus jannaschii TATA box-binding protein (TBP). Acta
Crystallogr. Sect. D Biol. Crystallogr. 60, 2328–2331
17 Grohmann, D., Klose, D., Fielden, D. and Werner, F. (2011) FRET
(fluorescence resonance energy transfer) sheds light on transcription.
Biochem. Soc. Trans. 39, 122–127
18 Bergqvist, S., O’Brien, R. and Ladbury, J.E. (2001) Site-specific cation
binding mediates TATA binding protein–DNA interaction from a
hyperthermophilic archaeon. Biochemistry 40, 2419–2425
19 Delgadillo, R.F., Whittington, J.E., Parkhurst, L.K. and Parkhurst, L.J. (2009)
The TATA-binding protein core domain in solution variably bends TATA
sequences via a three-step binding mechanism. Biochemistry 48,
1801–1809
20 Thomsen, J., De Biase, A., Kaczanowski, S., Macario, A.J., Thomm, M.,
Zielenkiewicz, P., MacColl, R. and Conway de Macario, E. (2001) The
basal transcription factors TBP and TFB from the mesophilic archaeon
Methanosarcina mazeii: structure and conformational changes upon
interaction with stress-gene promoters. J. Mol. Biol. 309, 589–603
21 Bell, S.D., Kosa, P.L., Sigler, P.B. and Jackson, S.P. (1999) Orientation of
the transcription preinitiation complex in archaea. Proc. Natl. Acad. Sci.
U.S.A. 96, 13662–13667
22 Adachi, N., Senda, M., Natsume, R., Senda, T. and Horikoshi, M. (2008)
Crystal structure of Methanococcus jannaschii TATA box-binding protein.
Genes Cells 13, 1127–1140
23 Littlefield, O., Korkhin, Y. and Sigler, P.B. (1999) The structural basis for
the oriented assembly of a TBP/TFB/promoter complex. Proc. Natl.
Acad. Sci. U.S.A. 96, 13668–13673
24 Kosa, P.F., Ghosh, G., DeDecker, B.S. and Sigler, P.B. (1997) The 2.1-Å
crystal structure of an archaeal preinitiation complex: TATA-box-binding
protein/transcription factor (II)B core/TATA-box. Proc. Natl. Acad. Sci.
U.S.A. 94, 6042–6047
25 Tsai, F.T. and Sigler, P.B. (2000) Structural basis of preinitiation complex
assembly on human pol II promoters. EMBO J. 19, 25–36
26 Selvin, P.R. (2000) The renaissance of fluorescence resonance energy
transfer. Nat. Struct. Biol. 7, 730–734
27 Ha, T., Enderle, T., Ogletree, D.F., Chemla, D.S., Selvin, P.R. and Weiss, S.
(1996) Probing the interaction between two single molecules:
fluorescence resonance energy transfer between a single donor and a
single acceptor. Proc. Natl. Acad. Sci. U.S.A. 93, 6264–6268
28 Tinnefeld, P. and Sauer, M. (2005) Branching out of single-molecule
fluorescence spectroscopy: challenges for chemistry and influence on
biology. Angew Chem. Int. Ed. 44, 2642–2671
29 Masters, K.M., Parkhurst, K.M., Daugherty, M.A. and Parkhurst, L.J. (2003)
Native human TATA-binding protein simultaneously binds and bends
promoter DNA without a slow isomerization step or TFIIB requirement. J.
Biol. Chem. 278, 31685–31690
30 Parkhurst, K.M., Richards, R.M., Brenowitz, M. and Parkhurst, L.J. (1999)
Intermediate species possessing bent DNA are present along the
pathway to formation of a final TBP–TATA complex. J. Mol. Biol. 289,
1327–1341
31 Whittington, J.E., Delgadillo, R.F., Attebury, T.J., Parkhurst, L.K., Daugherty,
M.A. and Parkhurst, L.J. (2008) TATA-binding protein recognition and
bending of a consensus promoter are protein species dependent.
Biochemistry 47, 7264–7273
32 Hieb, A.R., Halsey, W.A., Betterton, M.D., Perkins, T.T., Kugel, J.F. and
Goodrich, J.A. (2007) TFIIA changes the conformation of the DNA in
TBP/TATA complexes and increases their kinetic stability. J. Mol. Biol.
372, 619–632
33 Binnig, G., Quate, C.F. and Gerber, C. (1986) Atomic force microscope.
Phys. Rev. Lett. 56, 930–933
34 Hansma, H.G., Sinsheimer, R.L., Li, M.Q. and Hansma, P.K. (1992) Atomic
force microscopy of single- and double-stranded DNA. Nucleic Acids Res.
20, 3585–3590
35 Scipioni, A., Anselmi, C., Zuccheri, G., Samori, B. and De Santis, P. (2002)
Sequence-dependent DNA curvature and flexibility from scanning force
microscopy images. Biophys. J. 83, 2408–2418
36 Bussiek, M., Toth, K., Brun, N. and Langowski, J. (2005) DNA-loop
formation on nucleosomes shown by in situ scanning force microscopy
of supercoiled DNA. J. Mol. Biol. 345, 695–706
37 Dame, R.T., Wyman, C. and Goosen, N. (2000) H-NS mediated
compaction of DNA visualised by atomic force microscopy. Nucleic Acids
Res. 28, 3504–3510
38 Guthold, M., Zhu, X., Rivetti, C., Yang, G., Thomson, N.H., Kasas, S.,
Hansma, H.G., Smith, B., Hansma, P.K. and Bustamante, C. (1999) Direct
observation of one-dimensional diffusion and transcription by
Escherichia coli RNA polymerase. Biophys. J. 77, 2284–2294
39 Rivetti, C., Guthold, M. and Bustamante, C. (1999) Wrapping of DNA
around the E.coli RNA polymerase open promoter complex. EMBO J. 18,
4464–4475
40 Endo, M., Tatsumi, K., Terushima, K., Katsuda, Y., Hidaka, K., Harada, Y.
and Sugiyama, H. (2012) Direct visualization of the movement of a
single T7 RNA polymerase and transcription on a DNA nanostructure.
Angew Chem. Int. Ed. 51, 8778–8782
41 Milani, P., Marilley, M. and Rocca-Serra, J. (2007) TBP binding capacity of
the TATA box is associated with specific structural properties: AFM study
of the IL-2Rα gene promoter. Biochimie 89, 528–533
42 Milani, P., Marilley, M., Sanchez-Sevilla, A., Imbert, J., Vaillant, C., Argoul,
F., Egly, J.M., Rocca-Serra, J. and Arneodo, A. (2011) Mechanics of the
IL2RA gene activation revealed by modeling and atomic force
microscopy. PLoS ONE 6, e18811
43 Davis, N.A., Majee, S.S. and Kahn, J.D. (1999) TATA box DNA deformation
with and without the TATA box-binding protein. J. Mol. Biol. 291,
249–265
44 Kim, K. and Saleh, O.A. (2009) A high-resolution magnetic tweezer for
single-molecule measurements. Nucleic Acids Res. 37, e136
45 Tora, L. and Timmers, H.T. (2010) The TATA box regulates TATA-binding
protein (TBP) dynamics in vivo. Trends Biochem. Sci. 35, 309–314
46 Manghi, M., Tardin, C., Baglio, J., Rousseau, P., Salome, L. and
Destainville, N. (2010) Probing DNA conformational changes with high
temporal resolution by tethered particle motion. Phys. Biol. 7, 046003
47 Tolić-Nørrelykke, S.F., Rasmussen, M.B., Pavone, F.S., Berg-Sørensen, K.
and Oddershede, L.B. (2006) Stepwise bending of DNA by a single
TATA-box binding protein. Biophys. J. 90, 3694–3703
48 Schluesche, P., Stelzer, G., Piaia, E., Lamb, D.C. and Meisterernst, M.
(2007) NC2 mobilizes TBP on core promoter TATA boxes. Nat. Struct.
Mol. Biol. 14, 1196–1201
49 Gietl, A., Holzmeister, P., Grohmann, D. and Tinnefeld, P. (2012) DNA
origami as biocompatible surface to match single-molecule and
ensemble experiments. Nucleic Acids Res. 40, e110
50 Blair, R.H., Goodrich, J.A. and Kugel, J.F. (2012) Single-molecule
fluorescence resonance energy transfer shows uniformity in TATA
binding protein-induced DNA bending and heterogeneity in bending
kinetics. Biochemistry 51, 7444–7455
Received 30 October 2012
doi:10.1042/BST20120301
C The
C 2013 Biochemical Society
Authors Journal compilation 373