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The DNA Shape Code: From the
Angstrom to the Nanometer Scale
The average structure of dsDNA depends on the sequence: the
differences in the spatial arrangement imparted by the different base
pairs along the chain give rise to deterministic modulations of the
relative orientations of the average planes of the base pairs. These
orientations are commonly expressed in terms of the base-step
orientation parameters: roll, tilt, and twist.
(from B. Samorì and G. Zuccheri “DNA Codes for Nanoscience” Angew. Chem. Int.
Ed. 2005)
A code for the self-assembling of DNA single
strands in the atomic scale: the base pairing code
“the specific base pairing immediately suggests a possible copying mechanism for
the genetic material”
(Watson and Crick, 1953)
THE CODE OF THE DNA BASE PAIRING AND
SEMICONSERVATIVE REPLICATION
The code of the direct read-out in the atomic
scale for DNA-protein recognition.
Proteins involved in the regulation of the gene expression through their
interaction and binding to DNA control elements.
These proteins recognize
their specific binding sites
by sampling the specific
contacts
TATA-box binding proteins
Structural Codes for DNA Recognition
in the Nanoscale: Shape and Flexibility
The base sequence of a DNA segment also encodes the
dynamics of the chain. DNA is continuously morphing into
shapes and structures alternative to the canonical B-form, it
is coiling in the cell nucleus, it is “swilling lazily around in a
nourishing molecular soup of transcription factors and other
regulatory proteins that are milling around the nucleus.”
(from B. Samorì and G. Zuccheri “DNA Codes for Nanoscience” Angew. Chem. Int.
Ed. 2005)
How can we describe the DNA shape in the
nanoscale?
Eulero: sei gradi di libertà per descrivere
la posizione di un mattone rispetto ad
un altro (3 traslazioni + 3 rotazioni)
Solo 3 utilizzati dai gradini dinucleotidici: gli altri 3 bloccati dalle
interazioni idrofobiche + fosfati
Twist locale può essere
diverso dal twist globale
+20° ~ -10°
+ se le basi si aprono dalla
parte del solco minore
+ sopra a destra
+2Å ~ -1 Å bloccato dalle
catene fosforilossidriche
DNA Structural Parameters in the
nanoscale
La sequenza crea irregolarità nella struttura
della doppia elica
La sequenza induce curvature locali con una
specifica orientazione rotazionale e controlla la
flessibilità assiale della catena
Certe sequenze hanno mobilità elettroforetica anomala
Mobilità
Forma!
Varie molecole sono state costruite con la stessa sequenza con mobilità anomala,
mediante permutazione circolare, in modo che un tratto potesse essere ad
esempio in una molecola ad una estremità, in un’altra al centro, in un’altra all’altra
estremità
Come creare una curvatura su un piano, come in un marciapiede in giardino
Con 12 lastre giro di 180°
Angolo fra le lastre =15°
Raggio di curvatura= 12/2π =1.9 m
Se ogni lastra è larga 0.5 m
Come creare una curvatura in una pila di strati come nel
DNA: devo introdurre dei roll o dei tilt
Roll or tilt angles give rise to local bending
of the double-helix axis. These local bends
might lead to a zigzag pattern of the chain
axis, which remains essentially straight,
unless the bend occurs in phase with the
doublestranded helical repeat.
L’ introduzione dei roll o dei twist deve essere in fase con
l’avvolgimento dell’elica. Quattro soluzioni possibili
(a) Introduco un roll=45° ogni 10
coppie di ma qui entra molta
H2O.
Meglio frazionarlo
b) Roll=22.5° ogni 5 coppie di basi
ma devo alternare i segni: Il segno
del roll cambia perché dopo 5 c.b.
l’elica è ruotata di 180° e devo
cambiare segno per avere
curvatura sullo stesso piano
c) Congiungo 5c.b. con roll=14° a
5c.b. con roll=0. La piegatura
sembra comparire alla alla
giunzione come nei casi dei
tratti AAAA
d) Distribuzione continua del roll
gli angoli di roll per avere una
curvatura di 45° ogni 10 cb.
variano come la funzione coseno
Rn= 9° cos (Tw. n)
dove n=0, 1, 2,….; Tw= 36°
R0= 9°; R1=7.3°; R2=2.78°; R3= -2.78°; R4= - 7.3°
Posizioni ad alto roll e a basso roll
R5= -9° R6= -7.3° ……
Ma questi angoli di roll non contribuiscono pienamente alla curvatura sullo stesso
piano. a causa della loro rotazione specifica (Tw. n)
Lo faranno solo le loro componenti su di esso ottenibili attraverso il relativo coseno
direttore, quindi i loro contributi saranno:
Cn= 9° cos (Tw. n). cos (Tw. n) = 9° cos2 (Tw. n)
C0= 9°; C1=5.9°; C2=0.9°; C3= 0.9°; C4= 5.9°
La curvatura globale dopo 10 c.b. è 45°
C5= 9°……..
I singoli valori di roll che contribuiscono alla
curvatura dipendono dalla sequenza
These values make it possible to easily predict the lowest
energy chain conformation of a molecule from its sequence.
Positive or negative roll or tilt angles give rise to local
bending of the double-helix axis. These local bends might
lead to a zigzag pattern of the chain axis, which remains
essentially straight, unless the bend occurs in phase with
the doublestranded helical repeat. In this latter case, the
bend might give rise to extended persistent curvatures that
propagate from the angstrm to the nanometer scale, like in
the case of the large scale curvature is the 211 base-pair
segment from the kinetoplast DNA of the Trypanosomatidae
Protozoan Crithidia fasciculata. This is the most highly
curved natural DNA known
The most highly curved DNA fragment
211 bp segment from the
kinetoplast DNA of the
Trypanosomatidae Protozoan
Crithidia fasciculata
Its curvature is due to a
periodical recurrence od Atracts appropiately phased
with the coiling of the double
helix
CTAGCGATGA
CGGCGTTACC
GCAGTTTACG
ACAATTAGGC
TAACCTTATG
GGAATTCGAG
CGAGGTTACT
GCCAAAAAAT
TAACAAAAAA
AATGGCCAAA
TAGAGTCGAC
CCCTGCTGAT
AGAAACTCAG
AGAGAGATGA
TTGTACATAT
TATCATACAC
CTCGCCCGGG
TTTTTGGAGC
GCCAAAAAAT
TAGCGAATTT
AACGCACTGA
CTGCAGCCCA
TGGTTCGCTG
AAGGTTCGTC
TAGGGTCTGC
TGTCGTTAGA
ATACGATTTA
GATCCGGCCT
CCGAAAACCA
AGCGAAAATA
CCCTGAATTT
AAATCAAAAT
ACCATTTCCG
CAACCAAACC
TTCAGTAAGC
ACGCGGCTAC
GGTGACACTA
AAAATTCCAA
CCCAAAATCA
CCCCGAAAAT
TAGGCGAAAA
CTGAACGTCG
GGGTGCGGAA
GACTCTGACG
CAGATGCTAC
AATTAATACA
TAGAATACAC
CCGAAAATCG
AGGAAAAATG
TGGCAAAAAT
AACCCCCGAA
CCGGATCCTC
Si possono notare vari A-tracts quasi perfettamente in fase. Come dimostrato per
primo da Jack Griffith con la microscopia elettronica, questa particolare sequenza
impartisce una curvatura tale a questo DNA che un tratto di meno di 200 bp può
formare strutture circolari.
In pratica:
Un tratto di DNA molto curvo è quasi planare: è il modo per sfruttare al
massimo i contributi di curvatura.
Per fare un DNA curvo sul piano, allora tutti i contributi che fanno deviare la
direzione dell’asse molecolare devono curvare nello stesso piano. Per
ottenere questo è fondamentale che i contributi siano in fase con
l’avvolgimento della doppia elica. In pratica questo significa che i centri
degli elementi (tratti di basi) che imprimono una deviazione dell’asse dell’elica
devono essere spaziati di 10.5 coppie di basi (o circa). Questo è quello che la
natura fa per creare molecole molto curve (esempio, (A4G6)n).
L’elemento di sequenza che più di ogni altro imprime una curvatura è l’A-tract,
un tratto di 4 o più adenine. Non si sa troppo bene se è il tratto stesso che è
curvo o se piuttosto questo tratto ha una sua struttura molto diversa da quella
del DNA “normale” tale che alla giunzione si crei una discontinuità che induce
la curvatura (un poly-dA è rettilineo).
Se la fasatura degli A-tracts è un po’ minore o maggiore della ripetizione
dell’elica, allora si crea una curvatura nello spazio (superelica con (A4G7)n). Se
la fasatura è un sottomultiplo della ripetizione dell’elica, deformazioni opposte
si cancellano e generano un DNA deformato localmente ma essenzialmente
rettilineo (ad esempio elementi con spaziatura di 5 basi lungo la catena).
La curvatura del DNA è un fattore molto importante nella
regolazione genica: promuove e regola il legame delle
proteine e aumenta la probabilità che tratti di sequenza
lontani sulla struttura primaria possano trovarsi in prossimità
spaziale ed interagire tra loro (o contemporaneamente con
una proteina).
2. La sequenza determina anche una
orientazione rotazionale nella curvatura e
flessibilità
AA, AT, AC hanno un solco minore più stretto ( roll negativo) di CG, CA,
TA, TG (roll positivo)
Thermal motion
is superimposed
to the lowest free
energy profile
Static + dynamic contributions: DNA
shape and the flexibility
The shape assumed in space and in time by a particular
DNA molecule can be analyzed in terms of the
superposition of the thermal fluctuations of the structure
and the intrinsic, lowest energy structure of a chain with
that sequence.
Imaging many DNA molecules in air:!
100 nm
ensembles of profiles of different molecules!
Curvature computed from the angular deflexions along the
chain in AFM images
DNA profiles are fitted by segmental chains within the AFM
resolution
A method to study the shape of DNA molecules:
the local curvature of the chain
negative curvature
positive curvature
reading direction
C( n) = Co ( n) + f ( n) = Co (n) + f (n)
C0(n): intrinsic curvature at position n !
<f(n)>: average value of the curvature fluctuation!
Under the assumption of the first order elasticity: <f(n)>=0!
C(n) = Co ( n)
The local flexibility evaluated from the
curvature dispersion
flexibility
f (n, m)
2
= C( n, m) − C(n, m)
2
3. La sequenza controlla la flessibilità assiale
the axial flexibility is thermodynamically
related to the melting temperature of a DNA
tract when a first-order elasticity is assumed.
This data can be easily obtained from the
sequence by averaging the formal melting
temperature assigned to each dinucleotide
step.
DNA Denaturing
The melting temperatures correlate with the staking energies
-16
stacking energy (Kcal/mol)
-14
GC
-12
AC/GT
GG/CC
-10
GA/TC
-8
AG/CT
AT
CG
TG/CA
-6
AA/TT
-4
-2
300
TA
320
340
melting
360
380
400
420
temperature
Correlation diagram between experimental melting temperatures [1] and theoretically
evaluated stacking energies of dinucleotide steps in B-DNA conformation. Total
stacking energies were obtained by quantum chemical calculations [2,3].!
[1] Gotoh, O.; Tagashira, Y. Biopolymers 1981, 20, 1033-1042. !
[2] Ornstein, R. L.; Rein, R.; Breen, D. L.; Macelroy, R. D. Biopolymers 1978, 17,
2341-2360.!
[3] Saenger, W. “Principles of nucleic acid structure”, 137-149, C. R. Cantor,
Editor,Springer-Verlag- NY!
G-G Base Stacking
from UV melting and calorimetric analysis ( more accurate than )
Decreasing stability: GC/CG = CG/GC > GG/CC > CA/GT = GT/CA = GA/CT =
CT/GA > AA/TT > AT/TA > TA/AT
Is the higher
stacking energy
of the CG steps
due to the 3 Hbonds between
the two bases
contrary to the
AT step with only
2 H bonds?
DNA denatura in EtOH (cala temp melting)
EtOH rafforza i legami idrogeno, ma riduce le forze idrofobiche
Which is the role
of the H
bonding ?
DNA Denaturing and Renaturing
Local curvature and flexibility along
a DNA chain is ruled by the local
sequence.
Which is the role played by local
DNA curvature and flexibility in the
recognition processes between
DNA and proteins?
DNA-protein direct and indirect recognition
DNA-binding proteins involved in the regulation
of the gene expression
The sampling of specific contact at the atomic
level of resolution (direct read-out) is normally
preceeded by much faster read-out in the
nanoscale of the local curvature and
flexibility of the DNA chain (indirect read-out)
Architectural DNA-binding proteins (histones and
histone-like proteins)
The specificity of their binding is strictly
related to the sequence-dependent
curvature and flexibility of the
DNAtracts involved (indirect read-out).
Direct and indirect read-out in DNA protein
recognitions
1)
2)
How the indirect reading of the sequence in the
nanoscale by the proteins can take place?
Bending waves?
Funziona questo meccanismo anche con la TATA box?
The interface between the bound DNA and saddle-like TBP protein
contains only a few hydrogen bonds:
most of the interactions are hydrophobic in nature
Minor groove view into
the opening of step T1/A1'-A2/
T2’ by Phe301, and Phe284 Two pairs of phenylalanine residues, one in
each structural domain of the TBP molecule,
kink the helix by wedging between the outermost
two base-pairs of the TATA element.
This produces two sharp kinks in the helix,
destacks the base-pairs and pulls them slightly
apart. Any loss of stacking energy would be
compensated by the extensive van der Waals’
interactions between bases and phenylalanine ring
minor groove view of base step
T7/A7’-A8/T8’. Phe193 and Phe210 open basepairs 7 and 8 making extensive
van der Waals’ contacts.
ROLL: The kinks at the outermost two T-A steps, produced by
intercalation of phenylalanine side-chains, now are reflected clearly
in a large positive Roll,~ 50° which indicates an opening of the
angle between base-pairs toward the minor groove and
compression of the major groove. A large Roll contribution to bending also is found at the center of
the TATA box. RISE:
The forcing apart of base-pairs also is evidenced by a
Rise of more than 6 A°at those same steps.
TILT:
Tilt is insignificant, being generally less than 4°. This is in agreement
with past experience that Tilt rarely plays a significant role in DNA
bending.
• TWIST:
Twist angles all along the TATA box octamer are much lower than
the 36°expected for undeformed B-DNA. • Indeed, the helical twist is zero at the central step, these two basepairs are stacked directly atop one another. • In the seven steps within the TATA box, the helix loses an aggregate
123°of Twist, or one-third of a helical turn. • At the two kinked T-A steps this unwinding of the double helix
allows base-pairs to pull apart and to admit the phenylalanine
rings.
Anticorrelation between Roll and Twist: Roll must be in-phase with the doublestranded helical repeat
it tends to bring individual Roll steps into
a common bending plane, thereby
producing a true bend rather than a writhe. Why a TATA box?
Why a TA step is so untwisted?
Why a TA step is so untwisted?
TA high-slide step
X-ray diffraction data
Because of the high
slide of the TA steps
A TA step is untwisted because of its slide
S=0
Tw= 36
A 2A° Slide
reduces the
Twist from 36° to
28°.
The
phosphoribosidic
junctions are not
so rigid
S=2 A°
Tw= 28°
SLIDE OF THE DIFFERENT STEPS
(El Hassan, Calladine, Endeavour 20(2) 1996)
The superhelical tension underwinds the DNA
Basepair mutations in the WT TATA box
83° corresponds to the TATA box curvature of the DNA in the crystal complex.
three WT conformers with 83° curvature from the MD trajectory. The 8 bp TATA box is colored in cyan. The arcs fitted to the helix axes and their centers of
curvature are shown in red.
Only 2~4% of the WT, 27T and 30T conformers already adopt a 83° bent
conformation (see fig 3)
The distributions of global magnitude and direction of curvature of the eight
base pairs comprising the TATA box of each simulated sequence
The vertical broken line
intercepts the horizontal axis at
83°, (which corresponds to the
TATA box curvature of the DNA
in the crystal complex)
27T: the average curvature
decreased from 55° to 40° 30T: has a curvature that resembles
that of WT 29A: the effect is more drastic, the
curvature reduced to about half (55°
to 27°) Curvature is toward the major groove,
opening the minor groove. Direction of curvature fluctuates more
in 27T and 29A
When these smaller magnitudes of
global curvature are combined with
increased fluctuations in their
directions,
the effective result is a diminished
global
curvature of these TATA box
sequences.
Rel. transcription
Exp. curvature
Th. flexibility
(N.B. Curvature
correlates with
flexibility)
Th. curvature
The curvature of WT, 27T, 30T, and 29A correlates with the relative
transcription
This correlation indicates that a low inherent curvature may create a
significant barrier to formation of a stable, transcriptionally active TBP–
TATA box complex, as for 29A. The curvature favors a more stable complex characterized by ease of induced fit
upon binding of TBP.
The DNA sequence encodes the
nanoscale properties, superstructures, and
recognition mechanisms that DNA
exhibits.
DNA can provide recognition processes
whose selectivity and stringency can be
modulated on different length scales, such
as in the direct and indirect read-out
mechanisms between DNA and proteins.
Looking at biology from a nanoscientific point of view
introduces us into the methodologies and the purposes that
nature follows in its “engineering” of complex systems. As
Horst Stormer says in his lectures: “nanoscale science is
raising the lid on the biggest LEGO of the universe.”
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