This relarela-tionship 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,
Trang 1Transient DNA ⁄ RNA-protein interactions
Francisco J Blanco1,2and Guillermo Montoya3
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 relarela-tionship 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-tion and experimental determinaidentifica-tion of proteinÆnucleic acid complexes Structural genomics initiatives target the low-hanging fruits, small globular proteins that can be easily expressed as soluble material in heterolo-gous systems An analoheterolo-gous endeavor for RNA mole-cules has not yet been initiated, very probably because
of the experimental difficulties [1] Indeed, preparing large amounts of homogeneous RNA for crystalliza-tion is not trivial In addicrystalliza-tion, RNA samples need careful manipulation, are notably difficult to crystal-lize, give poor contrast in cryo-electron microscopy, and suffer from severe signal overlap in NMR spectra Protein–protein interactions may also be transient and difficult to capture experimentally, but they have been intensively targeted on a large scale by means of com-plementary methods, such as yeast two-hybrid and
Keywords
DNA; dynamics; endonuclease; interaction;
nucleosome; protein; ribosome; RNA;
structure; transient
Correspondence
F J Blanco, Structural Biology Unit, CIC
bioGUNE, Parque Tecnolo´gico de Vizcaya,
48160 Derio, Spain
Fax: +34 9465 72502
Tel: +34 9465 72521
E-mail: fblanco@cicbiogune.es
(Received 23 November 2010, revised 17
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.
Trang 2tandem 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
PCNA belongs to the group of DNA sliding clamps,
which are multimeric toroidal-shaped structures that
encircle the DNA duplex and act as platforms for
rep-licative polymerases and other proteins These
proces-sivity factors enable the polymerases to add thousands
of bases per second without detaching from the
geno-mic template The crystal structure of the
homotrimer-ic yeast PCNA bound to a DNA duplex was recently
solved [5], but only a few of the bases were seen,
prob-ably because of the transient nature of the interaction
in solution (Fig 1) The recent assignment of the
NMR spectrum of the PCNA ring [6,7] provides the
basis for solution studies of its interactions with DNA
and other proteins [8,9] The yeast helicase
minichro-mosome maintenance complex forms part of the
prere-plicative complex, and has been recently reconstituted
and loaded onto dsDNA [10] Electron microscopy
shows a barrel of two head-to-head hexamers that
encircles a stretch of DNA of approximately 68 bp
and passively slides along the DNA duplex
Sliding on the DNA may be common to proteins
other than DNA clamps before they bind to their
spe-cific target sequences Both one-dimensional diffusion
of proteins on the DNA (sliding) and direct transfer
between distinct binding sites (translocation) would
accelerate the search process relative to the
diffusion-controlled association–dissociation mechanism (Fig 2)
in the presence of a huge background of nonspecific
DNA, as occurs in the nucleus of the cell, with an
esti-mated DNA concentration of 100 gÆL)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
C
koff,A
koff,B
kon,C
koff,C
kdt,CB
kdt,BC
Fig 2 Proteins find their binding sites in an ocean of DNA sequences Scheme of a nucleic acid-binding protein (gray ellipses) involved in four dynamic equilibria (double arrows) and one-dimen-sional diffusion on the DNA double helix (single arrows) Binding to sites A, B or C occurs with different affinities (different kinetic kon and k off rates) Exchange between sites A and B (located close together in the DNA sequence) can occur through association–dis-sociation–reassociation or via diffusion on the DNA Exchange between sites B and C (located far away in the DNA sequence but close enough in space to collide) can occur through association–dis-sociation–reassociation or via direct transfer (kdt) For the sake of clarity, some of the species participating in the individual equilibria are omitted.
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 (http://www.pymol.org).
Trang 3conformational 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
most important factor in the indirect readout as
opposed to the direct readout These terms
differenti-ate between the recognition mechanisms based on
details of the DNA structure facilitating protein
bind-ing (indirect) and specific amino acid–base contacts
(direct) A recent examination of proteinÆnucleic acid
structures has shown how minor groove narrowing
enhances the negative electrostatic potential of DNA
and forms an arginine-binding site that is widely used
in protein–nucleic acid recognition [19] Minor groove width is primarily DNA sequence-dependent (A-tracts tend to narrow the groove, whereas GC pairs tend to widen it), although the geometry observed in a given complex is probably the result of both intrinsic and protein-induced conformation effects
Interactions lost in translation The 2009 Nobel prize in chemistry for studies of the structure and function of the ribosome rewarded a long-term effort by several laboratories The ribosome was probably the first biomacromolecular machine to appear in the early stages of life, and performs its function in essentially the same way in the three king-doms The ribosome translates the three-base codons
of mRNAs into the amino acid sequence of the pro-teins encoded in the corresponding gene (Fig 3) Because of its size (about 2.5 MDa) and lack of sym-metry, it took a long period of sample preparation refinement and the use of modern diffraction instru-mentation and methodology to obtain the high-resolu-tion structure of the 70S ribosome [20] In the process,
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].
Trang 4by 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
Escherichia coli ribosome By unbiased hierarchical
classification of 2 000 000 images, 50 structures of the
ribosomal substates during translocation were refined,
and the trajectories of the two tRNAs as they move
through the ribosome were visualized with a resolution
in the 10–20-A˚ range [21] Translocation is the final
step in polypeptide chain elongation, and involves the
concerted movement of the tRNAs, the mRNA, and
the 30S subunit relative to the 50S one The authors
used a molecular system in which retrotranslocation
was the actual movement observed After addition of
tRNAfMetto ribosomes loaded with fMetVal-tRNAVal,
retrotranslocation occurred on a scale of several
min-utes, and the samples at different time points were
extracted and frozen for cryo-electron microscopy It
was found that, at physiological temperatures, no
dis-tinct 30S subunit could be outlined, as it existed in
dynamic equilibrium with a large number of
conforma-tional substates The emerging picture of the ribosome
during translocation is that of a machine that couples
spontaneous, thermally driven conformational changes
to directed movement
Transient interactions also occur in tRNA loading
Whereas each Xxx-tRNAXxx is loaded with the Xxx
amino acid corresponding to its anticodon by a specific
synthase, most bacteria and all archaeons lack
glutami-nyl-tRNAGlnsynthase They produce Gln-tRNAGln in
a two-step pathway: glutamylation of the tRNAGln(by
the same low-specificity enzyme that glutamylates the
tRNAGlu), 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 tRNAGln and
tRNAGlu(the second and third bases), whereas the
so-called tail domain of the amidotransferase recognizes
the outer corner of the tRNAGln (specifically for the
tRNAGln) The two enzymes bound to the tRNAGln
assume alternative conformations for the two
consecu-tive reactions The catalytic centers of the two enzymes
compete for the acceptor form of tRNAGln, and
there-fore cannot adopt their productive forms
simulta-neously Hinge polypeptide regions between the
catalytic and anticodon-binding domains of the syn-thase, and between the catalytic and tail domains of the amidotransferase, allow both enzymes to adopt the productive or the nonproductive forms cooperating in Gln-tRNAGln synthesis, with a low probability of releasing the intermediate Glu-tRNAGln species This
‘alternative conformation’ mechanism may be more common than expected in consecutive enzymatic reactions
The transient positioning of the nucleosome along the genome The compaction of DNA molecules inside the cells occurs by supercoiling and binding to specific proteins
A supercoiled DNA duplex can form a toroid (the nucleosome, as in eukaryotes) or a plectoneme (inter-wound, as in bacteria) This distinction is relevant not 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 exist and facilitate the bending of the DNA around the histone core
Translational positioning [25] is strongly influenced
by the spacing between nucleosomes, but this spacing
is variable, with linker DNA regions in the range of
Trang 510–90 bp, and a given nucleosome can invade its
neighbor’s territory [26] Recent reports [27,28] indicate
that sequence-dependent histone–DNA interactions
have a predominant influence on the measured
nucleo-some occupancy (average number of histone octamer
levels on a given DNA region in a population of cells)
but not on nucleosome positioning (the extent to
which each of the octamers of the population found in
that DNA region deviates from its consensus location)
[29–31] Thus, the nucleosomal pattern in DNA coding
regions observed in vivo is not determined by DNA
sequence preferences for octamer binding, but
primar-ily arises by statistical positioning from a barrier near
the promoter, and this barrier involves an unknown
aspect of transcriptional initiation by RNA
polymer-ase II [28] Nucleosomal assembly in vitro, however, is
sequence-dependent
Obtaining well-diffracting crystals of nucleosomes
was a difficult task, and was strongly dependent on the
DNA sequence All structures corresponded to
nucleo-somes assembled from purified histones and human
a-satellite DNA sequences, until very recently, when
two new crystal structures of nucleosomes containing
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]
In prokaryotes, the DNA is condensed with polyam-ines and proteins In enterobacteria, the histone-like nucleoid structuring proteins perform this role and reg-ulate gene expression in response to environmental changes The crystals of the oligomerization domain of histone-like nucleoid structuring proteins reveal an assembly of symmetry-related dimers into a superhelix, establishing a mechanism for the self-association [35] Although there is no structure for the DNA-binding domain, the superhelical assembly suggests the forma-tion of a complex with plectonemic DNA
Engineering and design of protein-nucleic acid interactions – lessons from endonucleases
Restriction endonucleases are the DNA protein bind-ers with the widest application in biotechnology and biomedicine [36,37], and large efforts are being invested in the identification of new nucleases or the modification of extant ones to give novel or improved DNA sequence specificities [38,39] Recent successful examples of redesigned protein–DNA interfaces illus-trate our increased ability to achieve these goals by the manipulation of the direct readout interactions [40] By use of the crystal structures of the complexes, these methods aim at optimizing the amino acids for affinity
Fig 4 Structure of the nucleosome with bound RCC1 proteins Ribbon diagram of the structure of the RCC1Ænucleosome core par-ticle complex assembled from Drosophila melanogaster RCC1, Xenopus laevis histones, and the Widom 601 DNA (Protein Data Bank entry 3MVD) In this view, the DNA superhelix axis lies hori-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 (http://www.pymol.com).
Trang 6at 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 proteinÆ
protein complexes, there is still little structural
infor-mation on proteinÆnucleic acid complexes, especially
for chromatin enzymes and factors The transient
nat-ure of many of the interactions is probably one of the
major difficulties in their identification, isolation, and
structural characterization MS is emerging as a potent
tool for the study of dynamic or heterogeneous
pro-teinÆnucleic acid complexes [46] Although not a
high-resolution structural technique, it has the bonus of
requiring very little material Small amounts are also
used in single-molecule techniques, which are becoming
a fruitful approach to answer specific questions,
besides providing spectacular demonstrations of our
prowess in manipulating and observing protein.nucleic
acid complexes Recent studies have addressed RNA
folding by a helicase [47], DNA transport [48], and
DNA polymerization [49]
Crystallography will continue to be the main
tech-nique for high-resolution studies Tomographic electron
microscopy can provide structures of proteinÆ
nucleic acid complexes inside the cell [50], and NMR
has the potential to do so [51] NMR is uniquely suited
to characterize folding–unfolding events that occur at
disordered regions of proteins that become structured
upon recognition of their target nucleic acids, and can
be usefully complemented by small-angle X-ray
scatter-ing [52,53] Intrinsically disordered proteins or protein
regions will increasingly be the focus of structural
stud-ies as regulators of molecular recognition processes [54]
Acknowledgements
The work in the authors’ laboratories is supported by
grants from the Ministerio de Ciencia e Innovacio´n
(CTQ2008-03115⁄ BQU to F J Blanco, and
BFU2008-01344 to G Montoya)
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