Conformation of nucleic acids 4.1 Introduction Theimportance ofthe role played by nuclei acids in biological phenomena is now unquestioned. The deoxyribonucleic acid (DNA) in every cel behaves asa “data bank’, and the information stored in it enables each cellular constituent to be synthesized, assembled and regulated. Ribonucleic acids (RNAS) are involved in the building of ribosomes ribosomal RNA oF FRNA ), in protein synthesis (transfer RNA or RNA), incarrying the genetic message’ (messenger RNA or MRNA)and in regulatory mechanisms small’ RNA). Viruses are primitive entities containing. either a DNA or an RNA, while viroids are merely RNA fragments The structural model of DNA proposed by Watson and Crick (1953a,b) was a i that of jand 15 incredible the semi- inadequate about the lems. The by recent sare only jon of the possible to mation of bacterial 4.4 THE DOUBLE-HELIK STRUCTURE It is a right-handed double helix formed from two antiparallel helical phos- phodiester chains: ¥ — 5’ chaining oceurs in one helix and 5’ ¥ chaining in the ‘other (Fig. 4.13). The pairing of complementary bases (A and T, G and C) (Fig, 4.14) leads to an identical geometry for the AT and GC pairs. The four pos sible pairs (AT, TA, GC, CG) can ogcur inany arrangement whatsoever along the double helix without any appreciable local distortion ‘The angle of rvst around the double-helix axis defines the rotation around this axis when passing from one pair of bases to the next, This is similar to the angle between two suecessive stairs ofa spiral (strictly, helial) staiease. The symmet elements and the local referenee axes are illustrated in Figs 4.15 and 4.16 “The angle of tlt defines the rotation of one base pair with respeet to the next around the dyad axis. The angle of rol characterizes the rotational movement of ‘one base pair with respect to the other around the C8—C6axisina way similar tothe laths in a venetian blind, Using the general criteria for the existence ofa double helix it is possible to deline {wo main types of structure, called B and A. with the characteristics listed in Table 4.2, Both structures have a precise crystallographic definition in accordance \ith the various parameters and the seven charueteristie angles given inthe table Infact, we must consider each of these structures as capable of generating a family by small variations in the geometrical parameters. The C structure discovered {b) GC pair 49 Figure 13 Thergh-hanseddoutie htc ON, Figure 4.34 Watson -Crck paieng. & ‘ad ans (a 2-0 pseudosynmeny 2x5) nthe plane fhe basos brings he N8-Ci bond onthe purine sera correspondence wih he NIC! bons onthe pyrmine side50 Figure 445 sincotre mo sands ofthe double hei are antiparallel 2043 symmatty occurs inte phosphodiester hain so Atany point fhe douse helix. aprosohate group on one stand wth evincical eoornates (=) ean be associated win aconeeponang ‘group on re other sane wi coorinates (9, Figure 4:16 4 erence rane assocated wth he double rete can oe ‘ete asialows: he estaxis along the ine rom C8purne) 0 (CB(pyriicne) tne second iste dyad axisand neti isalongaline paraelto the ax ofthe helixand passing though theintrsacon othe fest two coordinate MOLECULAR BIOPHYSICS Axis of heli Pyrimidine Angie of rl Angie of tit ‘more than 20 years ago ean, for example, be considered as a variant of B with: base pairs per turn and a torsion angle of 386°. Similarly, in the A structure, angles ofthe chain are not the same in DNA and RNA. In the latter, the type ‘double helix is the general rule forall double-stranded RNAS. ‘This description of DNAs in terms of three or four well-defined structures prevailed for some 20 years. Such ‘archetypes’ arise mainly from the interpret ‘of X-ray diffraction patterns of DNA fibres, stretched under wellefined tions of hydration and saline composition of the medium. ‘Two experimental approaches have obliged us to take another look at Ananalysis of patterns from the electrophoresis of citcular DNA subjected partial enzymatic digestion (eg. DNAsel) shows that the periodicity ofthe B in solution is not 10 base pairs per turn but about 10.4, The “canonical clearly arises from theeonstraints of packing thedoublchelixinto the DNA fi is also found that the periodicity depends on the composition as far as bases concerned, being smaller for DNA rich in AT pairs than DNA rich in GC 2. By synthesizing oligodeoxyribonucleotides with perf ined sequ they can be erystalized and their atomic structure determined accurat4.4 THE DOUBLE-HELIK STRUCTURE 51 “Table 4.2 Some properties of BONA, ACDNA and ANA Property BONA AONA ee right-handed gnthandes sugar C2-endo ‘C3-2n00 . base parsper turn 10 1 pen 3A 2k ‘al dstance between base cars 34h 264 torsion angle between base pars 26 Br itarge rear about" ‘ish f mater groove” 4d 24h wt of mineroroave* 608 110A. ; ance oP atom tomalcal as 93A 954 Fetaton anges (30g) BONA ANA ANA at) a =90 =80 Be) 136 an 15 te) 38 a 8 aw) 139 3 83 «6 =133 185 «7 a) 57 45 78 x 73 a 8 endure char, eicesby SBA lon tr evan de Weare pose something that is impossible from a fibre diagram. The results from these expe ‘ments profoundly modify the regular primitive image of the double helix, When. looked at more closely, the beauty of the harmonious Watson—Crick double helix, similar to that of the Leonardo da Vine staircase in the Chiteau de Chambord, is . replaced by the imperfection of a spiral staircase with irregular steps that are all askew, Moreover, erjtallzation of the hexamer CGCGCG produces a left | banded double hei, cae the Z form because ofthe igzag shape ofthe phon ae wiih 93 phodiester chain. see es i strctrs the the type A. iuctures has terpretation This form had in fact been found in solution many years previously on an alter= ‘ating poly-D(CG)D(CG) placed ina strong saline concentration (Pohl and Jovin, 1972) A change in the sign of the circular dichroism that was observed inthis case could be interpreted as a change in the chirality of the double helix. An X-ray ook at the Analysis of the structure (Wang ai, 1979) shows that te structural nits Formed. | j 442 The2 form | from mo bse pairs. The an form fo . which sink oa C2-endo saat Preserved, G on the other hand is sym and is linked to a C3. The sign of T depends on the direction of rotation, ic. that of the helix described by each edge. Itisa number forming an intrinsie property ofthe ribbon, but is not necessarily an integer. It has become a metric property instead of a topological property. T'may be changed by any deformation ofthe ribbon, and can be calc lated per section ofthe losed ribbon, so that we shall then have Tay + Tye+ Notealso that Tis independent of the direction of the path since the sense ofa helix isan intrinsic property Comparing Land T: 1. For a single closed curve such as the axis of the double helix, Land T are meaningless For a relaxed circular DNA. i one in which the axis ofthe ribbon describes 4a plane curve, with S000 base pairs and 10 base pairs per turn, we have L-= +500, T= +500, so that L~T=0. If the closed curve representing the axis of the helix does not lie in a plane, LT is not zero. ‘The writhing number Wis defined as LT. It isa geometrical property rather than a topological one. Consider closed curve witha given value for W. Ii, around this curve taken as axis, we construct a helical ribbon, it cannot take on just any a Figure 4.24 Coordinate sytem or ‘etintonetT 5960 o-f Right-handed toroid superhelix Figure 4.25 Equltrun bewweun a et handed toro and aright handed supernens MOLECULAR BIOPHYSICS value for T since L must be an integer. If, for example, W= 1.7, then T= -2.7 (L=—Nor P= 1.7 (L=0)0r T=0.7(L= +0) ‘A few examples will make the meaning of LT id Weare: If we form, with a closed circular ribbon, two superimposed loops, the twist, is very small (7=0), Since L= +2, we have W's 2 (lhe axis of the ribbon describes a helix) Consider the telephone cable that connects the handset to the base. The attachment ofthis cable at its two ends is equivalent to a certain topological constraint defined by a number L.= TW. At each instant the eable will take on values of T and JV compatible with the elasticity (modulus of rigidity and bending modulus) of the material from which itis made. Ifthe cable is rot extended (handset on the base) Tis small and W large: the axis of the cable describes a helix. When the handset isin use the cable is stretched: the twist T increases and IV decreases 3. More precisely. fora closed ribbon wound round a eylinder and describing & right-handed helix, we have T=Nsina where Vis the number of turns of the helix and a the angle between the tangent to the helix and a plane perpendicular to the axis ofthe helix In this w Nsin N(I-~sina) Ifo tends to zero (telephone cable unextended), W tends to N. Ifa inereases and tends to =/2 (extended cable), H’ tends to zero, For most of the supercoiled circular DNAS found in eels (plasmids or viral DNAssuch as that of SV40), the number Zisless than a certain value Ly that would characterize a molecule having the same total number of base pairs but relaxed. where no twisting constraint remained. Since L-< Ly, AL = L~ Lo is a negative ‘number: there is larger number of base pairs per turn and hence fewer turns of the helix in the closed circular form than in the relaxed form. Despite this con- straint, in a given medium the circular form will end to recover the value of the angle of torsion £2 between two base pairs by writhing on itself; The writhing ‘number IV in this ease wll therefore be negative and we can also say that circular DNA forms negative “supercoils’, These may either occur as toroidal shapes or as superhelices (Fig. 4.25), the two forms being in equilibrium. ‘Writhing reduces the torsional energy but introduces a curvature that increases with the bending energy. Equilibrium is established between the two deformations ‘so as Lo minimize the total mechanical energy, The relationship between AE= L--Ly. the W number and the change in twist AT can be written AL=W+ar (4) Since the twist energy is generally much greater than the bending energy, AT is small and 1 large. The sum of the two energies forms the free energy (WV. T), ‘which may be considered as potential energy stored in the molecular structure. Note that Loisan operational quantity (all constraints have been removed) ands, ro longer a topological invariant. Depending on the external conditions (tem- perature, medium. ionic strength, ete) the value of Ly may vary. In addition, the45 PROPERTIES OF CIRCULAR DNA 6 -27 {quantities T'and WY cannot at present be directly measured experimentally, Because of this, the quantity AL=L~Zo, called the mumber of supercoils r. is the one generally use. A specific variation in Lis also defined by the ribbon o=(L-L)/lo (42) known as the supercoil density, For most natural circular DNAs, «lies between =0,03and -0.09, Once thecirele isclosed, AL remains constant aslong asnobreak osical en {insingle oF double strands) ours as) {In ivo, the funetion of enzymes known as topoisomerases isto alter the value of ews the linking number L. They can cut either one DNA strand (lopoisomerases 1) or a {wo posomerases 1). They thenallow apart ofthe DNA to passthrough the cu ess and re-stablsh the bond hetween the freeends. ATP isnot required inany ofthese processes. Topoisomerases I change L by +1 and topoisomerases Il change it by “+2. Inboth eases theresa reduction in the numberof supercoilsand ifthe process bing a continues we end up with the relaxed form. On the other hand, to introduce negativesupercols eto reduce Lincomparison with Z. there isanother enzyme. gyrase which consumes ATP. In energy terms, the creation of negative supercoils wil favour Further unwinding of DNA and hence the replication. transcription sn the ‘and recombination peosesses. Any variation in AT willbe reflected by a change in In this Wie. bya change in the tertiary structure of the DNA. 452. Physical properties of circular DNA “The presence of supercoils in a circular DNA is easily detected by electron increases microscopy but no quantitative determination is possible because of the very ‘method itself, The projection ofa three-imensional abject on to a plane does not allow supercoilsto becounted. On the otherhand, thecompact form resulting from. high value of W has hydrodynamic behaviour different from that ofthe relaxed. form. The latter has a coefficient of fiction f (ratio of the frictional foree to the speed ofthe particle) greater than that ofthe supercoiled form, The electrophoretic ‘mobility U = gB'f (where q is a quantity proportional to the charge and E's the electric field) and the sedimentation constanss = mr if-wherem” isthe effective mass fof the molecule, will hetefore vary with Was shown in the curves of Fig. 4.26. In both cases, the mobility and the sedimentation constant haveminimum values when 1= 0. On the other hand, the trinsic viscosity (7), which varies asa power of the mean size of the molecule, is maximized when W’= 0. These methods were or viral uv 5 : Figure 26 Varatonsf oectophortc i Lo rrbity Uandsesmeraton conta w with Ww. ae62 MOLECULAR BIOPHYSICS used, for example, to follow the unwinding produced by an intercalating molecule such as ethidium bromide (ETB). The plane aromatic ring is sandwiched between two adjacent base paits so that the distance from each of the base pairs to the intercalated aromatic ringis almost dential tothe initial distance between the two ‘base pairs (3.4 A in the B form), This elongation ofthe double helix is accompanied bbyan unwinding (AO < 0) whose value depends on the type of aromatic molecule. For ethidium bromide, A = ~26", Hence, if ETB is progressively intercalated cular DNA initially having + negative supercoil, a relaxed form is obiained for a number N of intercalated molecules given by toa N=2ar/0 For r= 25 and AQ-= 26, we find = 346, The unwinding produced by the intercalation has removed the constraint by taking the DNA back to its relaxed state under experimental conditions. Iti precisely this intercalation of ETB that was the origin of the frst work on. circular DNA. Beyond 346 molecules, we ean continue to intercalate ETB and hence to unwind DNA. this time by creating a positive supercoiling (> 0). In accordance with theoretical predictions, it ean be shown experimentally that the first part of the process (IW — 0) iseasier to realize than the second (> 0) sincein the latter we progressively increase the total free energy of the DNA. 453. Gel electrophoresis ‘Thefirst hydrodynamic methods were soon replaced by simpler method requiring only small quantities of material: agarose gel eleetrophoresis Ithas become the ist choice of methods for studying covalently closed citcular DNAs, ‘One-dimensional electrophoresis Firstly, it becomeseasy to separate closed supercoiled DNA froma relaxed DNA (witha cut single strand): because oF its compact form, the former migrates more rapidly than the latter. The two species appear as two bands several centimetres apart. IFelectrophoress is then carried out alter reaction with topoisomerase I, several bands appear, corresponding toa family of ropoisomers, so called because they differ from each other only by their £ value (Fig. 4.27a), Their migration depends on the value of W’ This is because the resistance offered by the gel to ‘motion inthe electric Geld is smaller when Wis large although there is of coursea limiting value, Howeve! iven medium and at a given temperature, the change in I reflects that of L and itis assumed that there is a difference AL of 1 between two successive bands AAs was clearly shown by Depew and Wang (1975) and by Pulleyblank et al (1975) this Family of topoisomers reflects the very conditions of functioning ofthe topoisomerase. Between the cutting and the reclosure of one ofthe strands, thermal fluctuations will ead tox Gaussian distribution of Z values around mean valve Lo corresponding to the working conditions of the enzyme. On closure, = O and Ly = T- Since the twist number T becomes higher us the temperature falls, the mean value Zo will depend on the closing temperature, An analysis ofthe Gaussian distribution ofthe bands around Z thus makesit possible to obtain the value ofthe fre energy G- arising from the presence of supercoil With r = L—Lq.we find AG. = K+" fora circular DNA with \ base pairs, For one since Tis constant in a Py a ae ee a ee eee Abie Z.xed DNA ase I becas he gl t rk eras the An possible 29°C mare @i il | Mh | ails | A, Figures.27 Since both +/Nand AG. 'Warealmost constant, VK must beconstant,as confirmed by experiment. We find NK~ I100R7 ‘obtain someidea ofthe sensitivity of the “tool provided bya plasmid havin example, 4000 base pairs. consider a rise in temperature of 10°C. This isMOLECULAR BIOPHYSICS present no physical method (X-ray, NMR, spectroscopy) capable of measuring SOSsystem,fi such small angular variation, ‘must be under ‘The followi ‘Two-dimensional electrophores . ‘encountered. Two-dimensional electrophoresis was developed a few years ago in order to deal «quantitatively with the transition from a double helix to another form, ‘The kink Theagarose gel plate on which the sampleis placed is first subjected toan electric First concein field in a given buffer solution (e.g, 100mM of trishorate-EDTA at pH 8). In fasrumed to comparison with a plasmid having a cut in the single strand (relaxed form), other section 25.2 plasmidsmigratemore quickly asthe number of supercoils rincreasesandeachspot such bends 00 differs from the next by a change in Z of one unit. However, itis impossible 10 is much small distinguish plasmids with positive supercoils from those with negative supereoilsin (Richmond e! this way, In order todo this, a certain number » of negative supercoils are removed duce a localia by an intercalating agent such as chloroquine added in sufficient quantities. Plas- ‘mids with a number of negative supercoilsr = narein telaxed form; thosefor which ‘The loop + > nstill have negative supercoils; and those for which r 200mno Cc for example, by the torsional energy stored in the supercoiled circular DNAs, ‘The transition zone between the left-handed and right-handed helix is also sen- sitive to the S; endonuclease. The conformation of such a junetion is not known precisely The conformational changes (kink, loop. cruciform, Z form) are generally caused by constraints due to the medium (pH, ionic strength) or by the molecule itself (supercoiling energy), but they can also be triggered by the binding of specific ligandsto the DNA. In intercalarion. the changein conformation iscaused solely by ligand interaction, Intercalation A molecule wth a plane aromatic ing (Muoren, acridine, penanthridin, et) toaybeinteealated in DNA, ce. slipped between two base pairs Sine the distance tecwcen aromatic mucus s determined by van der Waals radi (o he order of 34A) twoagjacent bas parsareabout 6A from each othe. Ths elongation of the DNA molecule isaccompanied by a local unwinding anda change nthe sugar puckerng. All such local changes in conformation naturally require acurate Structural suds, Three- and four-stranded hel Alternating sequences of the (4TAC),(4AdG), type located in negative supercoiled plasmids placed in a medium of pH <7 take on an unusual tertiary structure. ‘Triple helices and a kink are formed in a DNA conformation called H-DNA, whose enistence has been demonstrated in vitro. Such alternating sequences are found forexamplein the human genomeabout every 150kb, The supercoils may be induced during transcription but at the moment there isno prot that triple helices exist in vito, Four-stranded structures have recently been discovered in oligonucleotide crystals, They provide a good model for telomeric DNA located at the end fof chromosomes containing a Gerich and a C-rich strand. These specialized sequences could play a major role in maintaining the stability and integrity of chromosomes, 46.2. Flexibilit Conformational isonly limited rigidity in thes charged phosph molecule, at leas around the thre require a certain that DNA cha long DNA mole cases, positive important role. has proved poss hundred ase the problem of Aexibiltyunes ‘because thei ‘The general te diynamic aspect linear rigid mo The study of DNA molecule eiven medium. order to akein double helix The worrmlik Anintresting states between rotation (or G Consider N cone (ying on) Fig. 429.74.6 POLYMORPHISM AND FLEXIBILITY OF DNA a 462 Flexibility Conformational studies of DNAs and their double-helx structure show that there ‘sonly limited range of anglesin the nucleotide chain: in other words. thereissome rigidity in the structure. If we add 10 this the Coulomb repulsion between the charged phosphate groups, we should describe DNA to begin with as fairly rigid molecule, at feast over small engths of theehain. Yet DNA exhibits polymorphism around the three A, Band families and the change from one form toanother does require a certain ‘plasticity inthe molecule. However, the real problem sin fact that DNA chains fold in so many ways. We have already pointed out how much a long DNA molecule must be folded insde a phage head ora chromosome. In both cases, positively charged molecules (polyamines, histones, protamines) play an important role. More recently, however, without the help of any other molecule it has proved possible to build closed DNA circles containing not many more than a hundred base pars, The small radius of curvature (of the order of S060 A) rises the problem of the Flexibility of the chain: itis difficult to understand such a high flexibility unless at certain pointsin the molecule, there are regions already curved lar DNAs, because oftheir sequence orat least some regions that are more flexible than others. is also sen- The general term flexibility thus embraces both static aspects (curvature) and not known <éynamie aspects related tothe existence ofa double helix departing from the ideal linear rigid model. generally ‘The study of flexibility must therefore begin with an overall look at the whole the molecule DNA molecule, which wil involve the general principles of polymer mechanicsina of specific given medium, A more detailed analysis on a local scale will then be undertaken in solely by order to takeinto account thesequence and any irregulartiesin the geometry ofthe double helix. The wormlike chain (continuous curvature) An interesting model hasbeen proposed by Kratky and Porod (1949) o describe all, ide, ete.) states between the two extreme models of the perfectly Nexible chain with free the distance rotation (or Gaussian chain) and the perfectly rigid rod-shaped chain. the order of| Consider N segments of length a each making a small angle # with the previous weation of cone (lying on a cone of vertical semi-angle @ around the previous segment ~ see in the sugar Fig. 4.29). The mean value (1) of the projection on the fitst segment of the DNA, Figure 4.29 Pinar model of Kian Pore (1949) (worn ke chan) consising of Vsegmentsoflength a making an angle 8 wih agjacert ‘eames inthis exampe, the projection othe vector ring he wo fends ontothe frst eget at the end specialized integrity of aeMOLECULAR BIOPHYSICS end-to-end distance is given by (i) =a oe = a(t -x8)/(1—x) (as) where x= cos The persistence length Lpis defined asthe limiting value of has Noe, Werthen have Lp=a/(1-x) (45) (As #0, the chain can be viewed as one for which Lp remains finite, ie. in which, 42-0, which amounts to introducing a continuous curvature). Since is small, cos = 1-6/2and Lp = 2a) 46) Note that the persistence length doesnot depend on thelength Lalong the curse, ie. Na, butisan intrinsic property ofthe polymer in given medium. Iis elated 10 the structureand the interactions and not othe molecular mass. Ineqn 44, weean replace x” = (1-6 2)” by exp( N62) and putting the total length ofthe chain L= Naand usingeqn 46, we obtain 2 = exp(-L/Le) and hence (Hi) = Loll ~ exp(—L/L] (a7) ‘As Loo, (h) Lp, which agrees with the definition. If, on the other hand, the chainis smallenough for L < Lp, weobiain (#) ~ L. Thisshort chain behaves ikea. rigid rod of length L. With this worm-fike model, we can thusaccount for both local rigidity and the Nexibility ofa sufficiently long chain, ‘Another comparison between this model and those describing the behaviour of | ‘conventional polymers can be obtained by calculating (i). Ifristhe vector joining the two ends, we have (i?) = (rr). Hence AUP) = dir-#) = 2(r- de) = 240) dL (48) and replacing (h) using eqn 4.7, we obtain (I?) by integration: Ww ate ft -exp(-t/te bl The integration can be carried out directly and leads to WP) = 2p{L ~ Lol) ~ exp(—L/L0)]} (49) ‘When £50, eqn 49 takes the simple form (?) =2LLp ‘The quantity 2Lp appears to be equal to the starstca! link in a Gaussian chain. equivalent to this infinite worm-like chain, When LO, the rigid behaviourFigure 4.3070 MOLECULAR BIOPHYSICS than chains. The magnitude of the optical phenomenon depends both on the orientation and on the intrinsic physical properties of the molecule. However, interpretation of the data is difficult because of the approximations made ia hydrodynamic models. Moreover, in order to use pulsed electric fields that are strong enough, measurements have to be made at low ionie strength, Electron microscopy [Another approach is to measure the lengths Land Zp directly for a molecule deposited on the grid ofan electron microscope (Fig. 4.30). Although measuring ‘L poses no problem, that of Lp requires measurements ofthe angle @ between Wo segments separated by a distance s counted along the chain by using the relation- ship (only valid in 3D): cost exp(~s/ Ly) “The method can be used at any ionic strength and is independent both of the polydispersity and of molecular aggregation, since each molecule is dealt with separately. On the other hand, artefacts may be present depending on the method used for depositing the DNA on the grid. If insulficient time is made available to re-establish conformational equilibrium (sudden freezing for example), we then ‘observe the projection on toa plane ofa three-dimensional structure in solution Fromall the results obtained by either of these two methods, the persistence length of DNA in I mM NaCl an be estimated as approximately 600 A. Flexibility of the worm-like chain Once the geometrical parameters of the chain have been defined (essentially the total length Land the persistence length Ly). we can study its dynamics, How does this model behave when thermal agitation occurs? As our intuition would predict, the energy required to bend the chain is proportional to the persistence length (sce Box 4.1) jie ja? putting 14? = gwih dimensions MLIT=2), ETO estes elie tesd toa cds ieee AG=(e/2) | (anja ds4.6 POLYMORPHISM AND FLEXIBILITY OF DNAR MOLECULAR BIOPHYSICS “The persistence length Lp varies as 1/T for a constant bending coefficient, and substituting the value for g in eqn 6in Box 4. gives AG = RTLpA}/2L au) (an expression first obtained by Landau and Lifschitz). ‘Measurements of flexibility ‘The DNA chain srigid overa short distance (comparable witha rod) and becomes exible over large distances, The persistence length Lp introduced by the wormrlike chain model determines an approximate boundary between the two types of ‘behaviour. Several methods are available for the measurement of Lp, all of which depend on a determination ofthe radius of gyration Rg, since it ean be shown for the wormlike model that Re 2LpL {1/6 ~ Ly/ 2+ 13/ (Lp/L\ll —exp(—L/Le)}} (4.12) For argemolecules ike DNA, Ly/Lis very small and we can use theapproximation Ri LyL 3. “The radius of gyration can be determined directly by light scattering or small- angle X-ray scattering, or more indireetly from hydrodynamic quantities like the translational Brownian diffusion coefficient (determined by inelastic scattering of light), the intrinsic viscosity or the decay of linear electri dichroism. 463. The double-strand to single-strand transition ‘Thermodynamic aspects Following the model of Zimm and Bragg (1959) (see section 3.5.3), we denote paired bases byl" (AT or AU and GO) and unpaired ones by 0". There are two contributions to the energy of formation of a base pair: one is from the hydrogen, bonds (4G;) and other is from the increase in the stacking energy when changing. from two bases to two stacked base pairs (AG;). The latter term is absent for the first base pair ereated after a sequence of unpaired bases. Keeping the notation 5 and as defined in Chapter 3, we then have exp[-(AG, + AG:)/ RT) (a3) xp(-AG,/RT) (414) “The factors thus equal toexp(AGy/RT).Since AG: generally hasa high negative value, 2 <1 and can then be considered as a nucleation parameter. However, & special feature of the double helix must be taken into aecount: the formation of oops’ when a disorganized regions located between two double-stranded regions. If base pairs are disrupted, the loop is formed from m = 24+ 1) nucleotides. It ‘can be shown that the difference in entropy between this loop and a free ehain with thesame numberof inksis(3R/2)In(j + |) provided the oopsarelargeenough and any effect from excluded volume can be neglected. Since § = Rin W (on a molar scale), the probability of closing the loop varies as (j++ 1)"*7. A more rigorous expression for small loops is ofthe form: (e+ (1-0) (4.15) where A and B accompanied by ‘which will then “The results For For long chai lation of the me helical regionm We ean therefo 4 Hence, using th (i) is 0: maxim If, forexample fon average ev pint, the rati = 100, In other wor formed on ay regions ofthe Heterogenei ‘The caleulati ‘between AT: example), the ‘various methe parametersot ‘example, we {introduce two nam =AGxy bein double helix Ifthe DN! have forthe} ‘or, putingkdenote are wo 4.6 POLYMORPHISM AND FLEXIBILITY OF DNA where 4 and B are constants. In both expressions, the closure of a loop is accompanied by a AS < 0, This additional entropy change must be included in o, Which will then be given by fe pGs/ RT) die) + The results for very lng chains are identical to those already given For log chains, itis also possible to apply this simple modeling to the cae lation ofthe mean number (i) ot hia repions ata given temperature, Since each Icical region must he-primed. it contributes tothe patton function afactore Weean therfore writ (i) =ainZ/OIna =nDIn/BIng =n Di) D247) Hence, using the valve of (th) = nas/ (1 — 5) + 4s]! (4.18) (i) is maximum for s= 1 and has the value Wax = 82/2004 (19) If, forexample, = 10-4, then (2) = n/200, which means that helieal region exists ‘on average every 200 base pairs when T= Ty (s= 1). Since (k) = 7/2 at this point, the ratio (&)/(h) defines a kind of mean length, In this example, (k)/(h o!7 100, Inother words, the molecule can be viewed during the ‘melting’ processas being Formed on average from sequences of 100 base pairs separated by denatured regions of the same length, Heterogeneity of base pairs ‘The calculation can be made more realistic by taking into account the difference between AT and GC, the finite length of sequences (in the case of RNAS, for example), the charges carried by the phosphate groups, ete. A description of the ‘various methods used lies outside the scope of this book. but the influence of some parameters on the helix~coll transition can becalculated much more simply. If, For example, we wish to take into account the two types of pairing AT and GC, weean introduce two parameters sq and sp instead of one, with the definitions exp(—AGaa/RT) and sw =exp(-AGun/RT) (4.20) =AGxy being the free-energy change when a base pair type X is added to a double helix terminated by a Y pair. Only two values are used by assuming that Gan + AGna = AGaa + AGnw (421) I the DNA consists of » pairs of type B (GC) and |-» pairs of ype A (AT). we hhave for the transition temperature YAGun + (1 = W)AGa4 = 0 (422) (oF, putting k= s9/sq! pink +Insy =0 (423) B 44 MOLECULAR BIOPHYSICS ‘Taking as a standard a polymer formed solely from AT pais that “melts’ at & temperature Tp, eqn 3.22.can be integrated, and using eqn 4.23 gives (AH/R\(L/Ts = 1/To) = vIn (424) which implies a linear relationship between 1/T and &. We may then write: AT in = Ty ~ Ty = RTTow nh MH (425) and since TT, varies only a litle, there isin practice a linear relationship between Ty and the fraction v of GC pairs. Experimentally, AT is found to be approximately 0.43°C per %GC pairs under normal conditions (0.1 M Na” and pH 7) Influence of charges and ions The breaking ot formation of a double helix implies an interaction between two negatively charged polyelectrolytes. If Gg, denotes the free eneray of the double strandand G,,that of a single strand in theabsenee of any Coulombinteraction, the quantity (426) ‘represents the changeiin free energy accompanying the melting (all quantities refer to one nucleotide), Since AG = AH - TAS. and since the change is favoured by a rise in temperature, A// is positive. The number of degrees of freedom of a single Strand is greater than that of a double helix and so AS is also positive. The ratio [AH AS defines the transition temperature T, for which AG = 0. In the double helix, however, each phosphate group experiences the electric potential reated by the other phosphate groups situated both in the same strand and in the other strand, Only the first type of Coulomb interaction occurs with the Single strand. A term AG, representing an electrostatic repulsive energy must therefore be added to the free energy of the double helix. The transition is thus characterized by a change in free energy AG’ given by AG =AG-AG. (427) ‘The new transition temperature Ty is thus Th, = AH - AG. AS = Ty ~ AG/AS (428) Since AG, and AS are both positive, this energy due to repulsion between the phosphate groups destabilizes the double helix. ‘AL very low ionie strengths (=10-*M), NG; is large enough for the DNA to be partly denatured at ordinary temperatures. Since AG, isa linear function of log (being the ionic strength), the same applies to Ty, and the variation of Ty with base content and with the ionic strength can be represented a Ty = Glog 1+ 0.43(%GC) + 81.5 (429) aaeT which agrees quite well with experimental results. Itisinteresting to note the orders ofmagnitudeof A ffand AS, Asan example, wefind foracertain DNA, permoleof hase pairs, that AM =Skeal and AS=24e.u. (entropy units), which gives T= 91°C. At room temperature (20°C) and under the same conditions, we have AG~ L.6kcal. This. telatively small stabilizing energy for the double helix but nevertheless the probability of finding denatured DNA at room temperature isis thus (a2) 4.6. POLYMORPHISM AND FLEXIBILITY OF DNA, negligible because of the cooperative effect. Far from Ty (eg. at room tempera- ture, the probability of finding an opened base pair at a given instant is therefore very small ‘A more detailed analysis of the phenomenon shows that the energies (other than electrostatic) vary considerably, depending on the relative arrangement ofthe four nucleotides, and that some sequences (of the AT/TA type) are less stable than others. Such a local heterogeneity in stability is taken into account in more elaborate theories of the ‘melting’ of a double helix. These predict that there are regions or domains of different stability throughout the length of the DNA ‘molecule. A rise in temperature is accompanied by the successive melting” of these domains inthe order oftheir stability, Such a suecession of organized regions and ‘open looped zones has in fact been observed by electron microscopy. Moreover, by combining a temperature rise in very small jumps AT with a very precise detection of changes in absorbance A, a curve of d4/d7 against T'can be ‘obtained and forms kind of detailed map ofthe stability ofa DNA. In some cases, itcan be compared with functional domains (see Fig. 4.31). It is also possible to detect regions of lower stability inside plasmids. By increasing the density of superhelies, oval ‘melting’ occurs at constant tempera ture. The stored energy i used loally to unwind the double helix. Unwinding ten base pairs causes approximately one negative supercoil to disappear, 464 B-Z transition isonly recently that Z forms of DNA have been discovered ina bacterial plasmid inwhich an alternating GC sequence had been inserted. This sequence changes into A TC) o 2 0 & @ © 2 a & aw Cy ner oro. 000 7 (°C) 5052 St 56 58 60 G2 ue SDD 5 Figure 4.31 DA sng prose (anearzed $v40 ater ECOR! cura) (@)inkgra cure - varaton ot absorbanceat 270m wth emperate: (o)demane cuve ~ potoraaidT against T Inboth cases tre temperate israisecin stops o£ 405°C andthe enor ‘ntheabsorbance slesthan 10"* Curve (0) can beregarded as a superpostion of Gaussian ouves reoresertng meting ofcomains wit cree GC comes (From Gaba, 1978 Reprinted rm Anaiyica! ‘Biochemistry wih the permission of ‘eademic Press)76 Figure 4.22 8-2 yarsiton na las wth an inserted GC seauance ‘Comparson ot we plasmas, £22 with ‘ne Broan p23 wh two errs (8) ‘The two-dmensional elecrophorete palm produced on the gay the two pasmigs smutaneousy, (0) rerreation ta open cies represent p22, lutorces represent 123, and nes a plasmid wih one sng Stand break Rcan be seenthat p23 needa greater numberof supecois than p22 o adostaZ sructure. The aration ccur a 167 n9Z2ana at 187inpZ3 (Fem Elson etal 1985 Reprnes tom Proceedings ofthe National Acadony of Sences, USAW the authors permission) MOLECULAR BIOPHYSICS '8Z form witha suiient density of supercoils. From a thermodynamic point of the factor View. the transition occurs when itis energetically preferable to change from B1oZ focm ated rather than to inerease the twisting and writhing energy of covalently closed t= cular DNA. Detection relies on the fact that the change from right-handed toa tis then po left-handed helix introduces mean twist of about 6 per base paie. Comparing the Parameters 54 curve of eletrophoresis spots corresponding tothe different topoisomers of two junction, Expe plasmids it can be seen that for the one containing the (CG), sequence there is @ are ove suudden break at a certain number of negative supereols (Fig. 432). The electro- a best-fit proce phoretic mobility of these eireular DNAs depends solely on H, bwo spots of the (Peck and Wa Same mobility having equal values of W= L.-T. A reduction AT in the twist is Junction (oro Unexpected compensated at constant W by a corresponding reduction in AL. However, one spot ina two-dimensional elecirophoretic pattern differs from the next by AL = 1. Il-forexample, them spot after the transition has migrated a distance equal to that of the (n~5)th spot, it means that AL = AT =~ 5 Two quantities characterize the BZ transition, similar to those introduced into the Zimm and Brags theory of the helix coll transition, increase in AG 1, If AGne is the free-energy change corresponding to the change of one base pair from the B form to the Z form, the parameter sis defined by $= exp(—-2AGnz/ RT) (430) the factor 2 arising from the Fact that the unit in the Z form consists of two base pairs 2. IAG; is the free energy required to create w BZ junction, the nucleation parameter @ is defined by 1 Re? ch ecocecscscéesescscecscEcGC T6 iY y ch GcacecsascscscEcEcTEGescEe Te4.6 POLYMORPHISM AND FLEXIBILITY OF DNA. the factor 2 this time arising from the existence of two junctions with a B. form at each end of the sequence that changes into the Z form. Itis then possible (see Box 4.2) to calculate A7'asa sum depending on the two parameters s and ¢ and a parameter b corresponding to the unwinding &€ the junction. Experimentally, » and the number of supercoils are varied (Fig. 4.33). Curves showing the variation of AT with LL are obtained and can be used, with, aa best-fit procedure, to calculate the three quantities AGy,. AG, and b.Itis found, (Peck and Wang, 1983) that AG = 0.33 keal mol Okeal mol! of | junction (or c= 2.4 « 10-*at room temperature) and = -0.4 turn per junction ‘Unexpectedly. it is founel that the increase in onic strength isaccompanied by an Increase in AGuz, whereas for an alternating poly-<(CG) the BZ transition is, n8 Figure 4.38 Vriston of wit nuroor wth he rumer of supercis dng the BZ tanston.(From Peck and Wang, 1983 Reprinted tom Proceedings of the ‘National Academy of Scences USAwih ‘he authors permission) MOLECULAR BIOPHYSICS 8 10 2 4 «1 18 2 2 2 ~l-Le) ‘only possible ata high ionie strength, Moreover, the processis very slow. In 0.1 M. NaClat 20°C, the relaxation time is ofthe order of an hour, 465. Formation of a cruciform structure A cruciform structure appearing in a plasmid produces a delay inthe one-dimen- sional electrophoretic migration. In two dimensions, a sudden shift of the topoi- somersis observed, asin thecase ofthe B ~ Z transition. Thisis because the change from the B form to a cruciform structure oecursabovea certain density of negative supercoils. His accompanied by unwinding, i.e. by AT < Oand henceby Ar < 0.10 thepalindromicsequenceislongenough, thecruciformstructureisthestableonefor 5 > 0.03, However, since the rate of formation is very slow, preheating at 65°C is, necessary to accelerate the change from the B form tothe cruciform structure. The latter is therefore present during the frst electrophoresis. The introduction of an intercalating agent before the second electrophoresis unwinds the DNA. reduces 5 ‘and destabilizes the cruciform structure, Thus, for @ topoisomer of given LL, there are two forms (with and without cruciform), whieh are separated inthe first electrophoresis but migrate together during the second. Thetransition oocursovera small interval ofr = L.~ Ly and thus appears to be highly cooperative. ‘An analysis of the results leads to a free-energy change of ITkeal mol”! unfa vourable to the formation ofa eruciform structure 4.66. Three-stranded and four-stranded helices AL an acidic pH (pH 4), a mixture of equal proportions of oligonucleotides (dT.dC), and (GG.dA), leads to the formation of a rriple helix. One strand {dT.dC"), where C* isthe protonated cytosine, becomes detached from a part of, the double helix and is accommodated in the major groove ofthe other part of the doulle helix Formed cause (Fig. 4.35). Ty binge between Ina crystal peptide nuclei resolution. De “The PNA: D of three bases the favou may be formed are present. A plasmid is use double helix supercoils. Th using two-dim4.6 POLYMORPHISM AND FLEXIBILITY OF ONA double helix (Fig. 4.34), The TAT and C*GC associations of the triple helix thus formed cause a Watson Crick and Hoogsteen pairing to occur simultaneously (Fig, 4.35). Two single-stranded regions appear with a flexible kink acting as a hinge between the double and triple helix conformations. Tha crystallographic study of a complex between a dsoxyoligopurine and Wo peptide nucleic acids (PNA) (Bett 1a, 1995). triple helix was obtained at 2.5 A resolution, Deoxyribose is C¥ endo inside an A helix but the it is that ofa B form, ‘The PNA;—DNA triplex in fact formsa previously unknown helix with 16 groups ‘oF three bases per turn and a large cavity around the axis, Ifthe Favourable alternating sequence is inserted into a plasmid, the triple helix, ‘may be formed ata neutral pH as ongasa sufficient number of negative supercoils are present, AS in transitions studied previously, the stress energy stored in the plasmid is used to create a new structure incorporating some unvvinding of the double helix (appearance of a single strand) and reducing the number of negative supercoils. The formation of a triple helix can therefore be detected and measured using two-dimensional electrophoresis. Geacasarcrerercr crorcte-T~ ¢ POULT LOL 1 —CCTGTCCAGAGAGAGAGAGAGAG-A- > y—rererereré te Teron oy é : ° / », AGAGAGAGAGAGAGAG-A-S, ia Hoye g=6 o H-ys ee 4 tt Ng Boao meerereg =F on p45} g Er859 R £ERxF a ir we we § $856 aeegiae gp RES 77> EAL 9 Figure 4.34 Tio!ypes ofp hal pending on whether they} othe 3(Hy5) mow ofmeTC sands used. Hoogsieen pats av indeated by dos when bey re between A and Tand by crosses wren they aro between Gand ‘otonatedC Bases nbola ypeindiate prelred poris of attack n trob-srandea H-DNA by chemical robes such as ethyoyrocarbonate for A, osmium oxide frTand rmathoxyanine or (From Htun and Daroor, 988, Roped wi exmisson tem Seence vol 243, pp 1571—Band with heauthors pexmigsion © 1989 American Associaton er he Advancement ot Soence) ssFigure 4.35 Pessiie coubiepaiing (Watson -Crek and Hoogeteen) in trite nla Figure 4.36 Spatel dsrbuton ol Gand Tracts inthe yal The ourT groups form oops pring the antparate! Ghoices molecule A, Gt~Gi2 sone sland, GI3~-G24 sinectnecinmatecue B(rotshown) th coneaponding numbering G20-G42 and.G3-G54 The eters Sane A neareach guanine lnacate syn and and contormatons respectvely:thenumbers are values ty those in brackets releing 1 the 8 fom. (rom Kang era, 1902 Reprise wh permission fom Nature, wo. 356, po. 126-31 and wth Pe authors permission. © 1992 Mactan Magazines Lis) MOLECULAR BIOPHYSICS 10at-T05) ae 62, = al A 616 7 pam m7 “RTD yman 120 ae Nh4.6 POLYMORPHISM AND FLEXIBILITY OF ONA. Evidence for four-stranded DNA structures has recently been revealed ina erys- tallographie study (Kang ea, 1992) ofthe synthetic oligonucleotide d(G.T,Gy). The crystal contains a complex of two G,T.Gs units held together by cyclic hydrogen bonding of four Gs (Figs 4.36 and 4,37). The four-G pairing was indced Found about 30 years ago in guanosine gels and in assemblies of poly(G) or poly(1) (Guschibauer 7 af., 1990). The sugar~ phosphate backbone has an alternating, syitanti conformation. Apart from modelling a telomeric structure, such a four stranded DNA could play a role as transient structures in sister chromatid exchange. 4.6.7. Intercalation Lerman (1961) proposed an entirely new model forthe interaction between DNA and certain moleeles with more than one aromatic rng (Fig. 4.38), The planar tmolvuk intercalated between two se pis, Thedstance beeen the molecule tndcach ofthe adjacent pas oughly thesameasthat between double heise pairs ie-34A forthe B form. The intercalation process thus causes an clongaton ofthe molecule and an untwistng ofthe hel! Cron—clar4 ‘This general pictures confirmed by a series of measurements in solution, The increase in length L ofthe molecule can be measured dvelly by electron micro- scopy and insti hy the scattering of ght and the Increase in he innse sisosty (7) of small iid DNA fragments (ine in thi case varies as. The measurements are roughly in aprecment. with relationship of the type {P= fel -+2F), where ris the ratio of the moar concentration of the nterelated Imokcles to tat ofthe nuckotdes, Measurment of the unwinding requires the Use of supercoiled closed circular DNA. The unwinding angle depends on the nature ofthe intercalating agent, i. the stacking enery between the planar ao- matic molecule and the adjacent base pis “The ntl model enabled a whole series of problems to be tackled. These were: ‘the calculation of the binding isotherm: «# the intercalation energy and the role of charges, hydration and supercoiling: 81 Figure 4.37 Oxganzatonolguanine basesinthe quadruples Meskeltonant ‘ecrondensytfourGbasesaracranm inedetherstiwopianesctne molecule Notethecyelcsysiemerhydrogenbends wh nrspecivelengtns. (Flom Kang 1a, 1982 Repretegwnpermsiontiom Nature v0.958,p0 126-Standwihne ‘authors permission © 1992Macmilan MagaanesLis}82 MOLECULAR BlaPHYSiCS % Ehidium brome shox butt Daunomyen Figure 4.38 Exanies of mercalaing molecues the exact geometry of the complex: ‘© the kineties of the process ‘We now examine each ofthese aspects, and we shall find that many structural and chemical properties of DNA are involved. Binding isotherm The calculation of the binding isotherm for an intercalating molecule will depend on an assumption: intercalation between the ith and (i++ Ith base pairs introduces su the (—1)than binding with ‘model develo the T's and 4 where Got binding const Theisother r/A against r) where ris the’ to the total m shows that r/ intercalating However. t “upwards (Fig ‘can beseen by ‘experiments for r/A=0,1 ‘meaningless. Energetics Finding thee From the D pairs and the the sugars am that contribu intercalation around the 18.2.2) by alrand le will pairs 4.6 POLYMORPHISM AND FLEXIBILITY OF DNA introduces such a structural distortion that the probability finterealation between the (-I)thand ith and between the (/+ I)thand (7+ 2)th becomes negligible. This binding with exclusion of neighbouring sites is easily caleulated from the matrix ‘model developed previously in connection with the helix—coil transition. Insteqdof the “I's and ‘0's denoting monomeric units that are organized into helices oF not organized, here they will denote an oceupied or vacant site respectively. The accommodation ofthe intercalating ligand by the polymer s thus represented by a sequence of 0's and “I's We then have to define the statistical weight of an occupied site with respect (0 that of a vacant site, in the same way as we defined the statistical weight s of a ‘monomer organized into a helix with respect to that included in a statistical chain. The change in free energy AG accompanying the intercalation can be written, AG) = AG) RT In (432) where AGo, the standard free-energy change, is ~RT In K, K being the intrinsic binding constant and A being the concentration ofthe free ligand. Hence: AG) = =RT In(KA) (433) ‘Theisotherm is calculated in Box 43 and gives the Seatchard isotherm (graph of | yA against r)as 1/4 = K-29 (=r) (434) where rs the ratio ofthe mean number of sites occupied by the intercalating agent to the total number of sites, ie. tthe total number of base pairs. Equation 4.34 shows that r/4 =O when r=0.5, in agreement with the main assumption of one intercalating molecule every two base pairs However, the isotherm is no longer a straight line but isa curve that is concave "upwards Fig. 4.39), Moreover, the slope at the origins no longer ~K but ~3K, as, can be seen by Finding the value of dA )/drforr =0. I istherefore possible, when. ‘experiments are limited to small values ofr, to obtain a quasi-linear isotherm that, for r/A=0, gives an interscetion with the horizontal axis at r= 1/3, which is ‘meaningless. Energetics Finding the energy balance favourable to intercalation isa complex matter. From the DNA viewpoint, thereisa loss of stacking energy between adjacent base pairs and the crossing of several potential barriers during the structural changes of the sugars and the phosphodiester chain. Moreover, the chains of water molecules that contribute othe stability othe DNA are highly disturbed atthe postion ofthe intercalation site. The inereased separation of the charged phosphate groups around the intercalation site locally reduces the charge parameter (see section 18.2.2) by altering the distribution of counter-ions. 83 sa4 MOLECULAR BIOPHYSICS. erally bead Lastly th4.6 POLYMORPHISM AND FLEXIBILITY OF DNA 1A For covalently closed circular DNAs or more complex organizations ike the supercoiling of DNA around a histone core in a nucleosome, the unwinding induced by the interealation modifies the topological constraint, Inthe presence of negative supercoils (sce section 3.5.2), interealation is favoured, but it is diss favoured if i creates positive supercoils. In both cases, the stored twisting and. bending energy will be respectively added to or subtracted from the intrinsic binding energy. From the viewpoint of the intercalating molecule, there is van der Waals inter- action with the base paits on both sides of it. The energy of this interaction will depend on the geometry ofthe intercalating molecule and on the various atomic groups substituted in the aromatic rings. I ill also depend. fora given nucleotide sequence XpY. on whether X isin the 3’ or the 8’ position (Fig. 4.40). ‘Most intercalating molecules have a positively charged group (generally a nitrogen atom). Such a cation wll beattracted to the immediate neighbourhood of the DNA, where its concentration may be much higher than in the rest of the solution. Moreover. there is competition between the intercalating cation and the ‘usual counter-ions (Na°, K* or Mg™), The binding constant will therefore gen- cally bea decreasing function of the ionic strength. Lastly, the hydration ofthe fre intercalating molecule isconsiderably modified, ‘because the Volume in which the van der Waals envelopes of the various aromatic ‘molecules (bases and interealating molecule) are stacked is largely hydrophobic. ‘These general reflections on the energetics of the process show how difficult iis toincludeall thefactorsand to derive general laws. The large numberof parameters, involved also explains some ofthe contradictory results on recognition specificity. i.e the preferential binding ofthe intercalating molecule to certain sequences. For example, 9-aminoacridine binds more strongly to CpG than t0 GpG. 85 we Deeg ete | | | ’ H86 MOLECULAR BIOPHYSICS. @ Ethidium (2) { mF Siam @) ® madman: Ethidium (2) tenes] i ¥ ‘Ethidium (2) PF (2) «) OO POO-0-9 Figure 4.40 Peston che etn a bromide molecule between tw base 5’ end Pais: (a) odoUpA ard (0) ogoCpG FomTéai et 1977 Pern rom i ‘€3-endo Jourat oF Molec tony with he coe PF (1) ()Protavin molecule between to CpG pais. (Fom Shien ett, 1980 Reprinted tom Nocoe Aes Research by ermesion of Oxo Unversity Press land wih he aurrs permission) end Send ‘ca-endo c2-endo Geometry Crystallization of complexes between intercalating molecules and dinucleotides, had to be achieved before the local ehanges of structure in DNA after intercak could be described in detail Figs4.41 and 4.42), Inmost cases, thereisanalternation, ‘of both types of sugar puckering, each characteristic ofthe B form and the A form,Figure 4.43 Varaion ofthe angle 8 wth the peeuderotation chase Pang amelnudo MOLECULAR BIOPHYSICS 170" 110° 90° 70° 120" | 180°} 240" (On the 5’ side the nucleotide conformation is C¥-endo, with a low value of y and angle > in g*: whereas on the 3’ side it adopts the C2-endo conformation. a high value of x and 7 in g”. The only twist angle showing any appreciable change is 3, which changes roma valueclose to 180” inthedimeralone toa valueclose 0220" in the intercalation complex. Thisis the change related to the stretching of the helix. ‘The observed unwinding, which varies from one intercalating molecule 10 another (26° for ethidium bromide. seems to arise mainly from the alternating sugar puckering. The increase inthe rigidity ofthe helix may be partly due to the sugar C3-endo transition, which reduces conformational fluctuations. In fact, as indicated in Fig. 443, the variation of the angle 6 with the phase P and the amplitude of the pseudorotation passes through a minimum near P= 0.i.e. for the CY -endo form. Intercalation kinetics, Many fast-reaction kinetics experiments (see seetion 9.8) have enabled the steps in the formation of the intercalation complex to be unravelled. Two theories are possible 1. eis assumed that ata given instant a structural change in the DNA creates ‘cavity’, ie. a large enough space between two base pairs for the planar aromatic molecule to “lodge’ there, We then have the following two-stage kinetic in whic intereal ‘medium the inte preform 2. The po ifsc one off in whi Note th conformat Figure 4od due of y and ation. a high Eehange is J, set0 220° in oF the helix, molecule t0 e alternating fly duc to the gs. In fact, as P and the 0... for the ithe stepsin [theories are DNA creates the planar ng 1wo-st 4.6 POLYMORPHISM AND FLEXIBILITY OF DNA 89 kinetic process: Peet PL in which P denotes DNA, P* the new open conformation and L the imercalating molecule, A local transformation (which will depend on the medium and the temperature but also on the sequence) is followed by the interealation step in which the aromatic molecule takes its place in the preformed cavity (Fig. 4.48), 2. The positively charged molecule is first positioned near the phosphate by a diffusion-controlled mechanism. The aromatic partis temporarily lodged in for the conformational Nuctuation, ereating ine ofthe grooves while waitin an intercalation site, In this ease, we have the following Kinetic process & 4 Pal . PL, PL; k in whieh PL, is the “external” complex and PL» the intercalation complex. Note that the rate constants ky and ky in the first process relate to the conformational change of the DNA. In the sovond process, they characterize a Figure 4.44 Strworodol ote opening betwen wo base pais (CpG) to netcalaea plana— 90 MOLECULAR BIOPHYSICS diffusional encounter, while the conformational change is implicated in the tran- sition from PL to PLa, While the fist step takes place on a time seale of a ‘microsecond, the second is much slower and the lifetime of the complex (1/k_2)isot the order of 10ms. study ofthe variation of reaction rates with temperature gives ‘an activation energy of about 1520 keal mol! : These two values are approximately those found by NMR for the exchange of imino protons, ie. those involved in the hydrogen bonds between base pairs. In ‘both cases theresa rotation of the basesbut, forthe intercalation, wemust assume a coupling between that motion and the variation in 9. Biological consequences of intercalation Crick and co-workers (Brenner ea, 1961) proposed a model for indirect muta genesis by intercalation of aeridine molecules. At each intercalation ste there is a frameshift wenerally leading to a non-functional protein. However, three inter- calation sites succeed each other. the reading frame is recovered. In these triple ‘mutants a functional protein can again be obtained as long as the modified part does not play an important role, We can thus ay that the study of the mutagenesis induced by intercalation was the first indication ofthe existence of a triplet code Streisinger (1966) proposed a model for this frameshift mutagenesis based on several experimental fuets: 1. There are regions in a gene much more susceptible to modifications than others; these are called hotspots and correspond to repetitive sequences. 2. Mutations by addition are more frequent than those by deletion. 3. Inall cases, DNA replication must take place, indicating that there isa ink ‘between mutation and the replication fork In treisinger’s model, wehave to assume the existence ata given moment of a break and a “gap” in the DNA sequence resulting from repair, reomibination or repli- cation. Ithislack ofa baseislocated inside or near a repetitive sequence ofthe same base, sliding or mispairing may occur, During the synthesis to fill he ‘gap’ in the sequence cither addition or deletion of one or more bases may occur. Therole ofthe intercalating molecules in this model would be to stabilize the mispairing by ‘becoming preferentially bound at their level and thus to stop their rapid migration along the chain, This does not rule out a role specific to each molecule, 47 Structure of ribonucleic acids 47.1 Modelling Since only one of the DNA strands is transcribed into RNA, the latter no longer exhibits the regular complementarity ofthe bases on each strand that allows very long double-helix structures to be formed. However, the phosphodiester chain of RNA can fold on itself and create double-helix regions separated by single- stranded loopsof varying sizes. Thedilferencesbetween the geometry andstructure of DNA and RNA are avcentuated still further by the replacement of deoxyribose byribose. Allthe regions of RNA with a double-helix structure take on the A form tnd the ribose has the C¥-endo conformation (see Fig. 4.6). Moreover, the 2-OH group can form a hydrogen bond with the O4' atom ofthe neighbouring ¥ ribose, Which stabilizes and stiffens the structure. With any A. G, U and C sequence in the chain, there is @ priori a tremendous variety of conformations with the Watson~Crick type pairings AU, GC and A G-U. Rule the base seq are only ft halix is thee Since the! ‘would oocu ‘must exist difference 2 negativeay studied for! bycreating| double heli the melting ‘ment. In ge Gongs AG and mis th ‘These sin when more numberof} cach base p represented clover-leaft A-form dot4.7. STRUCTURE OF RIBONUCLEIC ACIDS 9 the tran G—U. Rulesforconstructinga secondary structure therefore had to besought from ale of a the base sequence. The problem is immediately simpler than that of proteins: there disor are only four bases forming three types of complementary paits and the double ure gives helix is the only possible secondary structure Since the formation ofa loop is discouraged because of the entropy change that : change of ‘would occur (AS <0), while each new base pairing reduces the free energy. there pairs. In | ‘must exist a structure for which the free energy is a minimum. The free-energy stassume difference AG between the structured RNA and a random chain must have as negative value as possible. Both the initiation and propagation processes already studied for DNA have tobe present to builda double helix. For RNA. theinitiation by ereating the frst base pairalso givesrise toa fairly large loop. The stability of the = double bln and hens elongtion mechanian, vl depend oa comparison of a. theming points ofthe AU and GC region wth the temperature othe exper ee inet In gotera ican be shown (Tinoco el, 197; se Box 44) ha, at 2°C, fese triple AGaoogation = ~ 1-2 keal for AU, —2.4keal for GC and 0 for GU. Moreover, ified part AGeraiatioe 2.3RT[B~ 1.5In(m + 1)], where Bis a constant of the order of ~3 fspenis toda tonne of unpaied base in the op. os. That sapere were enprve ae fee reve by Tar 187) i on when more sutures had Been determined, making possible to increase the umber of prameter and t ake inc acount he immediate neighbourhood of ons th cach bse pit Flat models of RNA can then be ult n which the double lis a represented by a ladder and the loop by a flat curve. For tRNA, thisis the so-called ees. CTover-ea/mode ig 448) A maximo numberof GC or AU pairings caren ; A-form double-helix regions (stems ), and unusual types of nucleotide (YU, SMeG, eis a link ; 3 ba brenk Bos rei. b Acceptor stem fthe same | sy " Fin the ee ofthe e:: g:3 cos an 7 B21 ween vik Doop | eB tee oo | d es ate ue vf Fchain of Creatas: cas nyaegenordrg by single- 1 ceca ine terbary structure Dots ndeate firucure 5 a 7 Standard son Orc psig Dribose Bn THe atatlelgop _etnereengoertay be : friable op nturng stomihes endo ne Anticodon oop En —— trimer acceptor let OCA OH o ‘cated (Fom Quer an ch 96. ribose, Gm a8 Repinted wth permission tom Scence Va T8450 790-806 and wine enon | (Constant nucleotide authors’ permission. © 1976 American Cand Constant purine or pyrimicine Asocaton re Aaancamertt Soerce)2 Figure 4.46 Secondary suet of Ecol 16SANA. (Flom Ste ef, 198, eprnad tom Jura’ of Molec Biology wih te permission of Academic Press) MOLECULAR BIOPHYSICS ‘DU, ete.) are located in single-stranded regions forming hairpin structures. Some base pairs are always found in the same position when all the known (RNA structures are compared. In 16S ribosomal RNA, we find a complex mixture of loops and stems (Fig. 4.46). 47.2. Three-dimensional structure Ils obvious that plane constructions lke those above do not represent the true spatial structure of RNA. Just asin the assembly of a 3D model from a fat card- ‘board blank cut to shape, the planar arrangement has to be folded, stuck together ‘and thus built up into. three-dimensional structure. ILisimmediately obvious that sucha structure plays decisive role inthe function of RNAs if we simply consider the process of translation, Several RNAs play absolutely vital roles in biological processes. Messenger RNA, for instance, caries the nucleotide sequence to be translated. Its precursor, which migrates from the nucleus into the cytoplasm inside nucleoprotein com- plexes, must undergo a prior splicing involving particular tertiary structures.4.7. STRUCTURE OF RIBONUCLEIC ACIDS ‘Transfer RNA. through itsanticodon loop, makes a selection from variouscodons «and introduces the amino acidsitearriesinto the peptide chain under construction. Ribosomal RNAs, with bound ribosomal proteins, define a precise tertiary strueture of the ribosome and the positioning ofthe other two types of RNA. Apart from their rolein translation, we should mention the importance of RNA structuresin the morphology of viruses and in that of roids, theshor fragmentsof RNA responsible forseveral tropical plant diseases. Finally. ata later stage we shall bbe looking at the role of this three-dimensional structure in the enzyme-like ‘behaviour of certain RNAs ribozymes) nll these examples, we need 10 know the ae 8394 Figure 47 Terry srucue conespondng othe oversea! modelot Fig 445 Hygrogen bonds beween bases are snow ae cross-range 0g the cold rations othe phosphodiester ‘aktone, The olack rungs represent ryctogen bonds satitzng te teriary struc (Fom Qugey and Fich 1978 Feprnied win permission fom Science, vo 194, 796-808 anche ‘unos peemisson. ©1978 American [Associaton or the Advancement ot Ssoene) MOLECULAR BIOPHYSICS spatial structure of the RNA, So far, however, the only structures to have been ‘obtained are those fa few NAsand viral RNAs together with thatofaribozyme, ‘The tertiary {RNA structure was initially determined from X-ray diffraction patterns of tRNA" erystals (Kim er a, 1974; Robertus etal, 1974) and more recently on {RNA crystals (Moras et af, 1980). A’ comparison of this three- dimensional structure (Fig. 4.47) with the clover-leaf model enables us to under stand what forces are involved in the folding of a nucleotide chain containing, between 74 and 91 nucleotides. Hydrogen bonds play a decisive role in many 1. In double-helix regions (stems), the standard Watson—Crick pairing is observed except for the existence of G-U pairs (Fig. 4.48), In other regions, hydrogen bonds are found in inverse Hoogsten pairings between A and U (Fig. 4.48b) in triple helices and in junctions between bases and backbone (sugars or phosphates) or between two backbone atoms. 3. Some hydrogen bonds enable the chain to form a kink. For example, at the end of the ToC loop of tRNA", a sudden kink is produced due 10 a hhydrogen bond between N3 of uracil $5 (vU) and phosphate 58, and another between O2 of the ribose in residue 55 and nitrogen N7 of guanine 57. A similar situation occuts at the beginning of the anticodon loop, with a hydrogen bond between N3 of uracil 33 and phosphate 36, and another between ribose OY’ of residue 33 and N7 of adenine 35 (Fig. 4.48). In both ‘ses, the chain rotates through 180° in the space of three nucleotides. 4, Hydrogen bonds between the ribose 2-OH group and a neighbouring phosphate may also cteate a bulge, ie. push out one or more bases, like those forming the variable loop. This bond is generally associated with a ribose transition from the C¥-endo form to the C2-endo form. iyo Sem) Acceptor stem ‘We might lait | (fel4.7 STRUCTURE OF RIBONUCLEIC ACIDS Figure 4.48 (2) G-U pairing (s0-caled wobble pair) The arrows show the S/—-3'drectonin eacn sugar—phosphale sand (0) Reversed Hoogseen paring between U and A () Formation ofa suddenkink The ange a(w) othe phosphate P ors fom is usual value, hus ensuing the ‘olaion ohefolowingbonds Theelsavan der Waals bond between he phosphate. andthe Urge snc ahycrogen bond betweunP,. and NB ohurack We might have hoped that some general rules governing the folding could be deduced from other tertiary (RNA structures, enabling us to set up prediction Algorithms applicable to other types of RNA such as ribosomal RNA, ‘Using few such rues, but mostly making use of many topological constraints, three-dimensional model of 16S RNA as it might be inside the 308 ribosome has recently heen constructed. The choice of the foldingin the model was mainly guided by the location of ribosomal proteins determined by neutron diffraction after selective deuteration and by a determination of their binding sites deduced from ‘accessibility measurements. When we remember all the LRNA models proposed. before 1974, none of which corresponded to the structure determined by X-ray diffraction itissurely advisable to avait the true structureofa ribosome now being termined by various physical methods. “7 9596 MOLECULAR BIOPHYSICS Pseudoknotting Inthe 1970s, the. end ofa plant virus RNA was found to be recognized by enzymes specific for RNA (Pinck er a..1970).Itisonly fairly recently (Peijer wl, 1985)that a model has been proposed that shows a three-imensional structure of this. end resembling that of (RNA. ‘The mechanism is described in Fig. 4.49 for the Turnip Yellow Mosaic Virus (TYMY). The pairing between the three guanines (13-15) of stem and the three eytosines (25-27) of stem 2Teads toa spatial alignment of eight basepairs organized into an A-type double-helix fragment. This is only possible if the two single= stranded regions A(IO)C(1UC12) and U2C22U23)C 24) eross the helix and sive the illusion of a knot: hence the term psewdokno1 given to such a structure. If sucha modified three-dimensional structure of thisend ofthe RNA isconstructed and compared with that of a 1RNA (Fig. 4.50), there isa striking resemblance suflicent to “balMe” the enzyme. Such structures are envisaged in the splicing ‘mechanism, i¢, the elimination of introns in the mRNA precursor. Ribozymes ‘When studying the splicing mechanism of the 268 RNA in a protozoan (Tetra ‘hymna it was ound (Cech eta. 1981) that the RNA was cut without the help of an enzyme. The RNA itself plays the role of the enzyme by engaging in sell: ‘leavaige. The reaction schemeis given in Fig. 4.51 for typell(thereare fourdifferent types). The guanosine that triggers the processis only involved through the ¥-OH,, Which acts asa nucleophilic group ancl causes series of transesteriications, i. an © on © 2 uc 10 *Uccce—cice UcaGAAccA ‘acccuNCu Ap yaccc GaGacccu OM ‘cucu 1 Figure 4.49 Fok ofthe end of TYMI/ ANA: (a) nlcaton of palingsto be mace: (0) ype ot foting o achieve te paras (Fom Pj etal 1985 Reprted rom Nucie Acids Research by Petsson cf Oxford Univers Press and wi the authors permision) ‘exchange be released intn ‘catalytic act ‘The firste ‘mined (Pley produced wi stranded D?$5) that $3’ end e Virus pe three ganized single: dix and ture. I structed mblance splicing (Cetra: help of in sel ferent O11, sic.an 4.7. STRUCTURE OF RIBONUCLEIC ACIDS exchange between phosphodiester bonds without changing their number. The wages and eyclizations. This ed introns then subjected to a succession ofc catalytic activity is related to the tertiary structure of RNA. ‘Te first crystal structure ofa catalytie RNA molecule has recently been deter mined (Pley et ul, 1994) in the ease of'a hammerhead ribozyme. The erystdl was produced with complex between a 34-nucleotide RNA anda [3-nucleotidesingle Stranded DNA inhibitor, The later becomes a substrate forthe ribozyme when a @ Figure 4.50(0) 97