REVIEW ARTICLE
Transient DNA
⁄
RNA-protein interactions
Francisco J. Blanco
1,2
and Guillermo Montoya
3
1 Structural Biology Unit, CIC bioGUNE, Derio, Spain
2 IKERBASQUE, Basque Foundation for Science, Bilbao, Spain
3 Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
Introduction
Protein–nucleic acids interactions
and structural genomics
In their celebrated reports on the double helix, Watson
and Crick showed the strong structure–function rela-
tionship in the DNA molecule. This relationship is
more intricate in the case of RNA, because of its lar-
ger structural and functional diversity, and even more
so when we consider proteinÆnucleic acid complexes,
given the much larger diversity found in proteins.
Rapid genome-sequencing methods, large-scale gene
expression analysis and high-throughput structural
genomics projects have greatly augmented the number
of known biomacromolecular structures. Currently,
about 72 000 structures are deposited in the Protein
Data Bank, but only 3% are nucleic acids and about
4% are proteinÆnucleic acid complexes. It is difficult to
know whether these figures mirror the prevalence of
proteins and their complexes in the cell, or whether
they arise from the greater difficulties in the identifica-

February 2011, accepted 11 March 2011)
doi:10.1111/j.1742-4658.2011.08095.x
The great pace of biomolecular structure determination has provided a
plethora of protein structures, but not as many structures of nucleic acids
or of their complexes with proteins. The recognition of DNA and RNA
molecules by proteins may produce large and relatively stable assemblies
(such as the ribosome) or transient complexes (such as DNA clamps sliding
through the DNA). These transient interactions are most difficult to char-
acterize, but even in ‘stable’ complexes captured in crystal structures, the
dynamics of the whole or part of the assembly pose great technical difficul-
ties in understanding their function. The development and refinement of
powerful experimental and computational tools have made it possible to
learn a great deal about the relevance of these fleeting events for numerous
biological processes. We discuss here the most recent findings and the chal-
lenges that lie ahead in the quest for a better understanding of protein–
nucleic acid interactions.
Abbreviations
PCNA, proliferating cell nuclear antigen; RCC1, regulator of chromosome condensation 1.
FEBS Journal 278 (2011) 1643–1650 ª 2011 The Authors Journal compilation ª 2011 FEBS 1643
tandem affinity purification followed by MS. Still, in a
systematic exploration of protein complexes in the
yeast interactome by tandem affinity purification fol-
lowed by MS [2], the protein proliferating cell nuclear
antigen (PCNA) was not detected in any of the 589
purified complexes, despite being a very promiscuous
protein and an essential component of the replication
machinery [3,4].
Replication and transcription
regulation – proteins in search of their
sites on the nucleic acids

)1
[11]. Sliding
and translocation events involve transient interactions
that are difficult to observe and even more difficult to
quantify. Crystal structures sometimes provide hints
about these events, e.g. by the lack of electron density
of DNA or protein regions, or by the observation of
different conformations of amino acid side chains asso-
ciated with the nucleic acid. Analysis in solution by
NMR is a more powerful approach to characterize
these systems [12], allowing the study of the kinetics of
translocation [13,14], as well as the structures of tran-
sient, nonspecific complexes. For instance, the struc-
ture of the Lac repressor bound to a nonspecific
(low-affinity) DNA sequence suggests that binding is
primarily driven by electrostatics, as most of the pro-
tein–DNA interactions do not involve the bases, but
the phosphates and sugars of the DNA backbone [15].
Most of these interactions are preserved in the com-
plex with the specific sequence, but, in addition,
numerous interactions with the bases take place. When
the overall structures of the two complexes are com-
pared, neither the DNA nor the protein undergo large
A
B
C
k
off,A
k
on,A

Fig. 1. Structure of a PCNAÆDNA complex. Two views of the
homotrimeric yeast PCNA ring bound to a short DNA duplex, as
deposited in the Protein Data Bank (entry 3K4X). The three poly-
peptide chains are shown as ribbons of different colors, and the
DNA as an orange rod (backbone) and blue–green sticks (bases).
The figure was prepared with
PYMOL ().
Transient protein–DNA ⁄ RNA interactions F. J. Blanco and G. Montoya
1644 FEBS Journal 278 (2011) 1643–1650 ª 2011 The Authors Journal compilation ª 2011 FEBS
conformational changes, but a protein segment that is
disordered in solution becomes less flexible in the com-
plex with the low-affinity DNA, and structured into an
a-helix in the complex with the specific DNA. There-
fore, the large local conformational landscape that the
protein populates in solution is reduced upon DNA
binding, and much more so when it specifically binds
to its high-affinity sequence. The protein conforma-
tional landscape can be narrowed by small-molecule
allosteric effectors favoring efficient DNA binding, as
found for the transcriptional activator CAP [16], and
the landscape of free DNA includes transient confor-
mations whose relevance still needs to be investigated
[17]. Many transcription factors bind to thousands of
places in the genome, not necessarily located in proxi-
mal promoter regions, and dissociate very fast in vivo,
which may be relevant for long-range and combinato-
rial regulation of transcription [18].
Electrostatics plays a driving role in transient pro-
teinÆnucleic acid interactions, as well as in selecting
and stabilizing the specific ones. Indeed, it may be the

a wealth of information has been obtained about the
mechanism by which the ribosome attains its high level
of accuracy in translation, its catalytic triad (rRNA,
ribosomal protein, and the peptidyl-tRNA substrate),
and the mode of action of many antibiotics, enabling
the design of novel ones (for a brief review, see
the Nobel Foundation Scientific Background published
50S proteins
50S rRNA
tRNA, E-site
tRNA, P-site
tRNA, A-site
mRNA
30S proteins
30S rRNA
Fig. 3. Structure of the 70S ribosome. The structure of the ribosome of Thermus thermophilus (Protein Data Bank entries 1GIX and 1GIY) is
shown, with the rRNA molecules represented by thin coils, the tRNAs by spheres, the mRNA by a thick coil, and the proteins by ribbons. In
the two views shown, the 50S subunit is at the top and the 30S subunit is at the bottom. The figure was obtained from Proteopedia [55].
F. J. Blanco and G. Montoya Transient protein–DNA ⁄ RNA interactions
FEBS Journal 278 (2011) 1643–1650 ª 2011 The Authors Journal compilation ª 2011 FEBS 1645
by the Royal Swedish Academy of Sciences at http://
nobelprize.org/nobel_prizes/chemistry/laureates/2009/
cheadv09.pdf).
However, translation is a dynamic process, the ribo-
some is a highly dynamic machine, and the crystal
structures can provide only snapshots of intermediates
along the process. It will be very difficult to obtain
crystal structures of all representative states of the
ribosome in action, but a low-resolution picture has
emerged from time-resolved electron microscopy of the

Xxx
is loaded with the Xxx
amino acid corresponding to its anticodon by a specific
synthase, most bacteria and all archaeons lack glutami-
nyl-tRNA
Gln
synthase. They produce Gln-tRNA
Gln
in
a two-step pathway: glutamylation of the tRNA
Gln
(by
the same low-specificity enzyme that glutamylates the
tRNA
Glu
), and amidation by the corresponding amido-
transferase. The crystal structure of the ‘glutamine
transamidosome’ of Thermotoga maritima [22] shows
that the anticodon-binding domains of the synthase
recognizes the common features of tRNA
Gln
and
tRNA
Glu
(the second and third bases), whereas the so-
called tail domain of the amidotransferase recognizes
the outer corner of the tRNA
Gln
(specifically for the
tRNA

only for DNA packing, but also for transcription, rep-
lication, and repair. The reason for this distinction is
not only how accessible the DNA is, but also the twist-
ing degree (overtwisted in the nucleosome and under-
twisted in the plectoneme). However, it has been
argued that a common topology for bacterial and
eukaryotic DNA-based processes might exist, as the
ejection of a histone octamer would convert the nucle-
osome into a plectoneme [23].
The crystal structure of the nucleosome core particle
[24] shows a compact assembly of 147 bp wrapped
around a disk formed by an octamer of histone proteins
(two copies of each one of the four core histones). How-
ever, this picture is deceptively static, because, in the
chromatin, the nucleosome rotational and translational
positioning is not fixed. Nucleosome rotational posi-
tioning (or register) defines the orientation of the DNA
helix on the histone surface, and a 10-bp periodicity is
observed, reflecting a preference for sequences that face
inwards or outwards with respect to the histones and
optimize DNA bending. Analysis of the minor groove
width along the double helix in 35 high-resolution crys-
tal structures of nucleosomes identified a pattern of 14
minima corresponding to regions where the DNA bends
and has close contacts with histone arginine side chains
[19]. The analysis of DNA sequences bound in vivo by
yeast nucleosomes reveals a periodicity for A-tracts
three bases long, with an average of 10 A-tracts per
nucleosomal DNA. Thus, even though long A-tracts
tend to be excluded from the nucleosome [25], A-tracts

the strongest known histone octamer-binding sequence
have been reported [32,33]. This sequence is the
Widom 601 DNA, the de facto standard for in vitro
nucleosome reconstitution in chromatin biology
research because of its tight binding, but it is a syn-
thetic repetitive sequence that may not be the best rep-
resentative of real genomic sequences assembled into
nucleosomes. The two structures are very similar to the
former ones, but with increased DNA twisting and a
145-bp core particle instead of the canonical 147-bp
one. The increased twist occurs at two superhelical
regions, which are the same regions where some of the
histone–DNA contacts differ from those in the a-satel-
lite nucleosomes. Therefore, the structure of the nucleo-
some can adapt to small variations in DNA length.
One of these two structures also contains the protein
regulator of chromosome condensation 1 (RCC1, also
known as RanGEF or Ran guanine exchange factor),
with implications for nuclear transport and mitosis.
This structure is the first to show how a nonhistone
protein recognizes and binds to the nucleosome
(Fig. 4). It was found that arginines of the switchback
loop of RCC1 interact with an acidic patch on the
histone H2A–H2B dimer, whereas the DNA-binding
loop interacts with phosphates of the nucleosomal
DNA. These results are consistent with RCC1 being a
non-DNA-sequence specific chromatin factor. Interest-
ingly, the acidic patch on the nucleosome is the same as
that occupied by the histone H4 tail of a neighbor
nucleosome in the crystal lattice of the nucleosome [34].

zontally and parallel to the plane of the page. The two RCC1 mole-
cules are shown in pale yellow and in magenta. The DNA is
represented by an orange rod (backbone) and blue–green sticks
(bases), and histone H3, H4, H2A and H2B are shown in different
colors. The two RCC1 molecules undergo equivalent interactions
on each side of the nucleosome core particle. Prepared with
PYMOL
().
F. J. Blanco and G. Montoya Transient protein–DNA ⁄ RNA interactions
FEBS Journal 278 (2011) 1643–1650 ª 2011 The Authors Journal compilation ª 2011 FEBS 1647
at the DNA interface, but they are not efficient for
complexes in which indirect readout is dominant in
DNA sequence recognition [41]. An increase in the
number of the crystal structures of proteinÆDNA com-
plexes should help to overcome this limitation [42,43].
Challenges and the way ahead
Most structural studies are carried out not with full-
length proteins, but with fragments. The most frequent
reasons for this are the difficulty in producing large
amounts of homogeneous material of large proteins,
and the simplification of the system to facilitate crys-
tallization and ⁄ or analysis by other techniques. How-
ever, investing time and effort in preparing and
analyzing the full-length protein can be extremely
rewarding, as shown by the information obtained with
the tumor suppressor protein BRCA2 and its interac-
tion with DNA [44,45]. As compared with protein Æ
protein complexes, there is still little structural infor-
mation on proteinÆnucleic acid complexes, especially
for chromatin enzymes and factors. The transient nat-

n
(CTQ2008-03115 ⁄ BQU to F. J. Blanco, and BFU2008-
01344 to G. Montoya).
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