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history The double helix: a tale of two puckers © 2003 Nature Publishing Group http://www.nature.com/naturestructuralbiology Alexander Rich The era of the double helix began 50 years ago with publication of the Watson-Crick formulation1 and the fiber X-ray diffraction patterns from groups led by Maurice Wilkins2 and Rosalind Franklin3. Analysis of the diffraction pattern, especially the fibers of the hydrated B-form, could be immediately interpreted as consistent with a double helix: the weakness of the first-layer line and the virtual absence of the fourth-layer line clearly were consistent with two chains wrapping around each other with the phosphate groups on the outside. Wilkins and colleagues gradually refined the nature of the double-helical model that could give rise to the increasingly detailed diffraction patterns. However, the diffraction pattern could not ‘prove’ the structure of the molecule as there was too little information. The deoxyribose sugar ring contains five atoms; they cannot all lie in one plane, and at least one atom must be out of plane (Box 1). With the continued analysis of the fiber patterns, it became clear that the B-form contained a ring pucker in which the C2′ atom was out of plane on the same became clear that the normal conformation in the hydrated in vivo environment involved the C2′ endo sugar pucker of B-form DNA. In the original double helix formulation, Watson and Crick1 had indicated two hydrogen bonds between the guanine and cytosine residues. Shortly thereafter, an analysis by Pauling and Corey4 strongly suggested that it was likely there would be three hydrogen bonds. This hypothesis was confirmed experimentally in 1963 in a single crystal analysis of the 1:1 complex of a guanine and a cytosine derivative5. Does RNA form a double helix? Watson and Crick1 suggestDeoxyguanosine with attached phosphorous atoms is shown in the two major nucleic acid sugar puckers. The C2′ endo pucker (left) is found in B-DNA, whereas the C3′ endo pucker (right) is found in A-DNA or in RNA. ed that it was unlikely that The distance between successive phosphate groups is close to 7.0 Å in C2′ endo and shortens to 5.8–6 Å in the RNA could form their proC3′ endo pucker. Nucleic acids can convert from one pucker to the other, although it takes greater energy for posed structure because conversion of ribonucleotides. Carbon, large open circles; hydrogen, small open circles; nitrogen, cross hatched the added O2′ of the ribose circles; oxygen, black circles; and phosphorous, heavily outlined open circles. would produce prohibitive van der Waals crowding. Starting in 1954, attempts were made to study RNA fibers to see if they would also form a double helix. The results were ambiguous. Slightly oriented patterns could be obtained, but they all looked the same, independent of the base composition of the material6. A breakthrough came in 1956 when, together with David Davies, More complex and not answered at the side as the base (C2′ endo). Because of that we discovered that mixing polyuridylic time was the question of why there were pucker, the phosphate groups could be up acid (poly(U)) and polyadenylic acid two forms. What was the nature of the to ∼7 Å apart. Such an extended poly- (poly(A)) would form a double helix, as less-hydrated fibers that produced the nucleotide chain influenced the rotation indicated by a well-oriented fiber diffracbetter-oriented and crystalline A-form, around the axis of successive base pairs. tion pattern7. Unlike the DNA diffraction which could convert to the B-form? In Study of the more complex A-form led to patterns, there was no change in the patthose days a half-century ago, fiber dif- the conclusion that the C3′ atom was out tern with changing hydration, and the fraction was the only way such large, elon- of plane (C3′ endo). In that conformation first-layer line was stronger than the gated molecules could be studied. the phosphate groups were ∼5.8–6 Å second-layer line. In addition, the diameGenerally, the patterns revealed that the apart. Thus, the sugar phosphate back- ter of the molecule was greater than that material in the fibers was rotationally dis- bone was shortened, producing a double of B-DNA. Eventually, it was discovered ordered about the fiber axis. This was helix in which the base pairs were slightly that the double-stranded RNA molecule often at the molecular level in the hydrat- displaced from the center of the helix to adopted a conformation similar to the ed B-form and usually as elongated produce a flatter and somewhat wider A-form DNA, exclusively using a C3′ endo microcrystals in the drier A-form. helix. Air-dried fibers were used to pro- sugar pucker (Box 1). The reason for this Although the diffraction patterns were duce the A-type diffraction pattern. The adherence to the C3′ endo sugar pucker limited in resolution, they were certainly A conformation was stabilized by the becomes apparent upon looking at the consistent with the formulation. Over the smaller number of water molecules com- position of the additional oxygen that next several years, work by Maurice pared to the B conformation. It soon would be present in the C2′ position of BOX 1 Two major nucleic acid sugar puckers. nature structural biology • volume 10 number 4 • april 2003 247 © 2003 Nature Publishing Group http://www.nature.com/naturestructuralbiology history ribose. In the C3′ endo conformation there is adequate separation between the oxygen on C2′ and the oxygen on C3′, in contrast to a van der Waals crowding that occurs if the sugar pucker were C2′ endo. Because of the unfavorable energetic situation of ribose in the C2′ endo conformation, RNA molecules are usually found in the C3′ endo conformation. There is a significant energy barrier between the two puckers for ribose; in contrast, the deoxyribose ring has a small barrier. Finding that poly(A) reacted with poly(U) to form a double helix7 was surprising at the time. This was the discovery of the first hybridization reaction in which long polynucleotide chains formed a double helix based on the specificity of hydrogen bonds. Within a year, together with Gary Felsenfeld, we discovered that a second strand of poly(U) could be taken into the helix to make a triple helix of one poly(A) plus two poly(U)8. Because there was no increase in the diameter of the helix, we suggested that the additional uracil residue was bound by two hydrogen bonds to the amino group and N7 of adenine. This interpretation was considerably strengthened two years later by Hoogsteen’s singlecrystal analysis of 1-methyl thymine in complex with 9-methyl adenine9. The DNA–RNA hybrid helix The availability of short polynucleotides in the deoxy series chemically synthesized by Khorana and colleagues10 made it possible to ask whether a hybrid helix could be made with one RNA strand and one DNA strand. It was known at that time that the conformation of B-DNA in solution was quite different from that seen in the RNA–RNA duplexes; thus, it was not obvious that they could combine. In 1960, I could show that a two-stranded molecule would form with an RNA chain (poly(A)) and a DNA chain (polydeoxythymidylic acid or poly(dT))11. This reaction was important in two respects. It was the first DNA–RNA hybridization (which is still used today in isolating mRNA through their poly(A) tails), and it also represented a model for how RNA polymerase might make an RNA strand by forming a hybrid duplex with a single DNA strand. One year later a purified preparation of DNA-dependent RNA polymerase revealed that this was precisely how the enzyme worked12. It was only later that it was possible to show that in the hybrid helix the DNA molecule conformed to the RNA pucker so that both strands had the C3′ endo sugar pucker (Box 1). The important considera248 tion was that changing the conformation of the RNA strand was energetically costly, but changing the conformation of the DNA strand was relatively easy. This largely explained why the melting temperature of the RNA duplex was considerably higher than a DNA duplex with the same sequence, and the melting temperature of the RNA–DNA hybrid was intermediate. The double helix at atomic resolution A number of single crystal diffraction studies of purine-pyrimidine co-crystals were carried out in the 1960s. A disturbing trend was found in that all co-crystals of adenine derivatives with uracil or thymine derivatives had Hoogsteen pairing, but none had Watson-Crick base pairs13. This led to a Hoogsteen model of the DNA double helix14 that did not fit the diffraction data as well as the WatsonCrick model, but one could rely on the fiber diffraction data only to a limited extent. The question remained: what was the structure of a double helix? The first single crystal structures of a double helix were solved in my laboratory in 1973. This was before it was possible to obtain oligonucleotides in quantities suitable for crystallographic experiments. However, we succeeded in crystallizing and solving two dinucleoside phosphates, the RNA oligomers GpC15 and ApU16. Furthermore, the resolution of the diffraction pattern was 0.8 Å. At atomic resolution we could visualize not only the sugar phosphate backbone in the form of a double helix but also the position of ions and water molecules. By extending the structure using the symmetry of the two base pairs, it was possible to generate RNA double helices that were quite similar to the structures that had been deduced from studies of double-helical fibers of RNA. The GpC structure had the anticipated base pairs connected by three hydrogen bonds. However, the ApU structure showed for the first time that WatsonCrick base pairs were formed when the molecule was constrained in a double helix, as opposed to the Hoogsteen base pairs that were favored in single nucleotide complexes. The significance of the double helix at atomic resolution was recognized by the editors of Nature, who, in their News and Views commentary, called this the “missing link” and recognized “the many pearls offered” to help resolve one of the major uncertainties in nucleic acid structure (Nature 243, 114; 1973). These structures capped the effort that I had started some 20 years earlier which had been initiated in earnest with the recognition that poly(A) and poly(U) would form a double helix. High-resolution crystallographic analyses of larger fragments of the double helix (DNA or RNA) did not emerge for another six to seven years with the availability of chemically synthesized oligonucleotides. The complex manner in which the RNA double helix can fold into novel conformations was first visualized in the structure of tRNA. The rate-limiting step was obtaining a crystal of sufficient resolution. In 1971, we found that the addition of spermine to yeast tRNAPhe led to a crystal which diffracted to 2.3 Å resolution17. This was the first crystal of a macromolecular nucleic acid, and heavy atom derivatives as well as methods to identify the end of the molecule had yet to be discovered. This was before the days of area detectors or cryo-crystallography, and computer programs were also quite primitive. However, by 1973, at 4 Å resolution, the architecture of the polynucleotide chain could be traced through visualization of the electron dense phosphate groups. This revealed an unusual L-shaped structure in which double helical segments were organized to form the arms of the L18. The detailed nature of the complexity was visualized a year later at 3 Å resolution in which a number of interactions were found beyond the Watson-Crick base pairs in the double helical segments19,20. Although the nucleotides were predominantly in the C3′ endo ribose conformation, there were places where the chain had to span a longer distance. In one segment, the chain was lengthened through two nucleotides adopting the C2′ endo conformation. This was a good illustration of the manner in which an otherwise inflexible RNA strand would adopt the less favorable conformation locally to yield an overall stable structure. This field of complex RNA structure has now exploded with ribozymes and, most recently, the ribosome. Careful inspection of those structures reveals changes in sugar ring pucker at selected positions, usually outside of the double helical segments. DNA single crystals and left-handed Z-DNA The advent of DNA single crystal analysis awaited the development of chemical methods for synthesizing oligonucleotides. In the late 1970s these methods were becoming successful. At about nature structural biology • volume 10 number 4 • april 2003 © 2003 Nature Publishing Group http://www.nature.com/naturestructuralbiology history this time, I established a collaboration with a Dutch nucleotide chemist, Jacques van Boom, on a DNA single crystal study. Since G-C base pairs were more stable, I suggested a simple sequence d(CGC GCG) that might be induced to crystallize. To our delight, it crystallized and diffracted to 0.9 Å resolution. Here, at last, we could see the DNA duplex at atomic resolution. To our surprise, what emerged was an unusual left-handed form of the double helix: two antiparallel chains still held together by Watson-Crick base pairs but in an entirely different conformation than that seen for B-DNA21. It was a somewhat elongated and thinner molecule that had only one groove; it was related to right-handed B-DNA by flipping the bases upside down so that the upper surface of a base pair in B-DNA becomes the lower surface of the base pair when it changes to Z-DNA. This inversion results in rotating the purine bases about their C-N glycosyl bond (Box 1), so that it assumes the syn conformation. But most interesting was the fact that it also changed the pucker of the purine ribose ring so it adopted the C3′ endo pucker found in RNA molecules. In progressing along the polynucleotide chain the bases had an alternation of anti and syn conformations and also an alternation of C2′ endo and C3′ endo sugar puckers. The molecule was a surprising hybrid in terms of ring pucker. This led to a zig-zag backbone, hence the name Z-DNA. Although there had been indications of a conformational change in poly(dG-dC)22, we were quite unprepared for accommodating this alternative conformation into our picture of DNA biology. This conformation was unstable relative to B-DNA because the phosphate–phosphate distances were somewhat shorter, and the electrostatic repulsion is less in the B conformation. However, it was later found that negative supercoiling would stabilize the Z conformation23. This discovery led to the recognition of transient Z-DNA formation in biological systems in segments that were subjected to negative torsional strain, especially during transcription. Other sequences crystallized as B-DNA, the most important of which was the Dickerson dodecamer24. It served as the basis for many different studies of the effect of a sequence on conformation. More recently, the development of NMR methods for solution studies has made it possible to define the structure of oligonucleotides free of lattice constraints. This has greatly enhanced our knowledge of the structural biology of DNA molecules. Today, a great many complex DNA structures are visualized, including fourstranded conformations. Many DNA– protein structures are seen, such as the intricate folding of B-DNA around the nucleosome core, studies of DNA undergoing replication or transcription or with significant changes in topology as, for example, due to the TATA box–binding protein. In all of these studies, attention is paid to the ring pucker of the DNA molecule since changes from the simple, unperturbed double-helical B-DNA con- nature structural biology • volume 10 number 4 • april 2003 formation are usually associated with changes in the pucker, utilizing the elastic element inherent in the polynucleotide backbone. Alexander Rich is in the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. Tel: 617-253-4715; Fax: 617253-8699. 1. Watson, J.D. & Crick, F.H.C. Nature 171, 737–738 (1953). 2. Wilkins, M.H.F. & Randall, J.T. Nature 171, 738–740 (1953). 3. Franklin, R.E. & Gosling, R.G. Nature 171, 740–741 (1953). 4. Pauling, L. & Corey, R.B. Arch. Biochem. Biophys. 65, 164–181 (1956). 5. Sobell, H.M., Tomita, K. & Rich, A. Proc. Natl. Acad. Sci. USA 49, 885–892 (1963). 6. Rich A. & Watson, J.D. Proc. Natl. Acad. Sci. USA 40, 759–764 (1954). 7. Rich A. & Davies, D.R. J. Am. Chem. Soc. 78, 3548 (1956). 8. Felsenfeld, G., Davies, D.R. & Rich, A. J. Am. Chem. Soc. 79, 2023–2024 (1957). 9. Hoogsteen, K. Acta Crystallogr. 12, 822–823 (1959). 10. Tener, G.M., Khorana, H.G., Markham, R. & Pol, E.H. J. Am. Chem. Soc. 80, 6223–6230 (1958). 11. Rich, A. Proc. Natl. Acad. Sci. USA 46, 1044–1053 (1960). 12. Furth, J.J., Hurwitz, J. & Goldmann, M. Biochem. Biophys. Res. Commun. 4, 362–367 (1961). 13. Voet, D. & Rich, A. Prog. Nucleic Acid Res. Mol. Biol. 10, 183–265 (1970). 14. Arnott, S., Wilkins, M.H.F., Hamilton, L.D. & Langridge, R. J. Mol. Biol. 11, 391–402 (1965). 15. Day, R.O., Seeman, N.C. Rosenberg, J.M. & Rich, A. Proc. Natl. Acad. Sci. USA 70, 849–853 (1973). 16. Rosenberg, J.M. et al. Nature 243, 150–154 (1973). 17. Kim, S.-H., Quigley, G., Suddath, F.L. & Rich, A. Proc. Natl. Acad. Sci. USA 68, 841–845 (1971). 18. Kim, S.-H. et al. Science 179, 285–288 (1973). 19. Kim, S.-H. et al. Science 185, 435–439 (1974). 20. Robertus, J.D. et al. Nature 250, 546–551 (1974). 21. Wang, A.H.-J. et al. Nature 282, 680–686 (1979). 22. Pohl, F.M. & Jovin, T.M. J. Mol. Biol. 67, 375–396 (1972). 23. Peck, L.J., Nordheim, A., Rich, A. & Wang, J.C. Proc. Natl. Acad. Sci. USA 79, 4560–4564 (1982). 24. Wing, R. et al. Nature 287, 755–758 (1980). 249