Lecture (2): Genetic Implications of DNA Structure PDF

## Lecture (2): Genetic Implications of DNA Structure - After Oswald Avery and his colleagues demonstrated that the transforming principle is DNA, it was clear that the genotype resides within the chemical structure of DNA. - Watson and Crick’s great contribution was their elucidation of the genoty...

## Lecture (2): Genetic Implications of DNA Structure - After Oswald Avery and his colleagues demonstrated that the transforming principle is DNA, it was clear that the genotype resides within the chemical structure of DNA. - Watson and Crick’s great contribution was their elucidation of the genotype’s chemical structure, making it possible for geneticists to begin to examine genes directly, instead of looking only at the phenotypic consequences of gene action. - Determining the structure of DNA permitted the birth of molecular genetics—the study of the chemical and molecular nature of genetic information. - Watson and Crick's structure did more than just create the potential for molecular genetic studies; it was an immediate source of insight into key genetic processes. Three fundamental properties of the genetic material were identified. - First, it must be capable of carrying large amounts of information; so it must vary in structure. Watson and Crick's model suggested that genetic instructions are encoded in the base sequence, the only variable part of the molecule. The sequence of the four bases adenine, guanine, cytosine, and thymine along the helix encodes the information that ultimately determines the phenotype. Watson and Crick were not sure how the base sequence of DNA determined the phenotype, but their structure clearly indicated that the genetic instructions were encoded in the bases. - A second necessary property of genetic material is its ability to replicate faithfully. The complementary polynucleotide strands of DNA make this replication possible. - Watson and Crick wrote, "It has not escaped our attention that the specific base pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” - They proposed that, in replication, the two polynucleotide strands unzip, breaking the weak hydrogen bonds between the two strands, and each strand serves as a template on which a new strand is synthesized. The specificity of the base pairing means that only one possible sequence of bases—the complementary sequence—can be synthesized from the question of how DNA, with only half a dozen components, could act as the genetic information was answered by James Watson and Francis Crick in 1953. - Their now famous double helix provided a chemical basis for the genetic code and suggested a mechanism for DNA replication. - The structure of the DNA double helix by James Watson and Francis Crick Like the bases of DNA, Watson and Crick formed a complementary pair. - **Structural model for double helix DNA.** ### Watson and Crick in the 1950s - Figure (1):James Watson (b.1928) and Francis Crick (b.1916), with their model of part of a DNA molecule in 1953. - In 1950 Maurice Wilkins and his assistant Raymond Gosling took the first images of DNA using X-ray diffraction. - Gosling’s work was continued by Rosalind Franklin who joined Wilkins’ group the following year. - Linus Pauling, placed the phosphate backbone of DNA down the middle, so failing to solve the structure. - The data proving the phosphate backbone was on the outside of the double helix came from Rosalind Franklin, an X-ray crystallographer at London University. - Watson and Crick used a X-ray diffraction picture taken by Rosalind Franklin and Raymond Gosling in 1952 as the basis for their structural model. Rosalind Franklin (died in 1958 of cancer aged 37, probably due to the effects of the X-rays). - Unraveling the chemical basis for inheritance won Watson, Crick and Wilkins the Nobel Prize in Physiology or Medicine for 1962 “for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material." - This central finding underlies our whole understanding of how living cells operate and what life means. - Since the discovery of the double helix, the genetic code has been worked out, and starting in 1995 with the bacterium Haemophilus influenzae, the DNA of a variety of organisms has been totally sequenced. ### Native DNA Is a Double Helix of Complementary Antiparallel Strands - The modern era of molecular biology began in 1953 when James D. Watson and Francis H. C. Crick proposed that DNA has a double-helical structure. - Their proposal was based on analysis of x-ray diffraction patterns of DNA fibers generated by Rosalind Franklin and Maurice Wilkins, which showed that the structure was helical, and analyses of the base composition of DNA from multiple organisms by Erwin Chargaff and colleagues. - Chargaff’s studies revealed that while the base composition of DNA (percentages of A, T, G, and C) varies greatly between distantly related organisms, the percentage of A always equals the percentage of T, and the percentage of G always equals the percentage of C, in all organisms. - Based on these discoveries and the structures of the four nucleotides, Watson and Crick performed careful molecular model building, proposing a double helix, with A always hydrogen-bonded to T and G always hydrogen-bonded to C at the axis of the double helix, as the structure of DNA. - The Watson and Crick model proved correct and paved the way for our modern understanding of how DNA functions as the genetic material. - Today the most accurate models for DNA structure come from high-resolution x-ray diffraction studies of crystals of DNA, made possible by the chemical synthesis of large amounts of short DNA molecules of uniform length and sequence that are amenable to crystallization (Figure 2a). - DNA consists of two associated polynucleotide strands that wind together to form a double helix. The two sugar phosphate backbones are on the outside of the double helix, and the bases project into the interior. - The adjoining bases in each strand stack on top of one another in parallel planes (Figure 2a). - The orientation of the two strands is antiparallel; that is, their 5’-3’ directions are opposite. - The strands are held in precise register by formation of base pairs between the two strands: A is paired with T through two hydrogen bonds; G is paired with C through three hydrogen bonds (Figure 2b). - This base pair complementarity is a consequence of the size, shape, and chemical composition of the bases. - The presence of thousands of such hydrogen bonds in a DNA molecule contributes greatly to the stability of the double helix. - Hydrophobic and van der Waals interactions between the stacked adjacent base pairs further stabilize the double helical structure. - In natural DNA, A always hydrogen-bonds with T and G with C, forming A-T and GC base pairs (Figure 2b). - These associations, always between a larger purine and a smaller pyrimidine, are often called Watson-Crick base pairs. - Two polynucleotide strands, or regions thereof, in which all the nucleotides form such base pairs are said to be complementary. - However, in theory and in synthetic DNAs, other base pairs can form. - For example, guanine (a purine) could theoretically form hydrogen bonds with thymine (a pyrimidine), causing only a minor distortion in the helix. - The space available in the helix would also allow pairing between the two pyrimidines cytosine and thymine. - Although the nonstandard G-T and C-T base pairs are not normally found in DNA. - Nonstandard base pairs do not occur naturally in double-stranded (duplex) DNA because the DNA copying enzyme, does not permit them. - The two strands of DNA are held together by hydrogen bonds. A hydrogen bond is longer (approximately 0.3 nm) and are much weaker, with A G values in the range of -10 to -30 kJ mol-1, while Covalent bonds are short (0.095 nm) and are very strong, with ΔG values in the range of -100 to -500kJ mol-lAs found in the double helix, adenine forms two hydrogen bonds with thymine, and cytosine forms three hydrogen bonds with guanine. - A single hydrogen bond is itself very weak, the 2500 hydrogen bonds that hold together every kilobase of DNA provide an extraordinary level of stability to the helix. - The AT base pair is less stable in thermodynamic terms than a GC base pair. - Most DNA in cells takes the form of a right-handed helix. The x-ray diffraction pattern of DNA indicates that the stacked bases are regularly spaced 0.34 nm apart along the helix axis. - The helix makes a complete turn every 3.4 to 3.6 nm, depending on the sequence; thus there are about 10-10.5 base pairs per turn. - This helical form, referred to as the B form of DNA, is the normal form present in most DNA stretches in cells. - On the outside of the helix, the spaces between the intertwined strands form two helical grooves of different widths, described as the major groove and the minor groove (Figure la). - As a consequence, the atoms on the edges of each base within these grooves are accessible from outside the helix, forming two types of binding surfaces. DNA-binding proteins can “read” the sequence of bases in duplex DNA by contacting atoms in either the major or the minor grooves. - Under laboratory conditions in which most of the water is removed from DNA, the crystallographic structure of DNA changes to the A form, which is wider and shorter than B-form DNA, with a wider and deeper major groove and a narrower and shallower minor groove (Figure 3). - RNA-DNA and RNA-RNA helices also exist in this form in cells and in vitro. - Figure (2): The DNA double helix. - Space-filling model of B DNA, the most common form of DNA in cells. The bases (light shades) project inward from the sugar-phosphate backbones (dark red and blue) of each strand, but their edges are accessible through major and minor grooves. - Arrows indicate the 5’-3’ direction of each strand. Hydrogen bonds between the bases are in the center of the structure. - The major and minor grooves are lined by potential hydrogen bond donors and acceptors (highlighted in yellow). - Chemical structure of DNA double helix. This extended schematic shows the two sugar-phosphate backbones and hydrogen bonding between the Watson-Crick base pairs. - Figure (3):Comparison of A-Form and B-Form DNA. - The sugarphosphate backbones of the two polynucleotide strands, which are on the outside in both structures, are shown in red and blue; the bases (lighter shades) are oriented inward. - The B form of DNA has about 10.5 base pairs per helical turn. Adjacent stacked base pairs are 0.34 nm apart. - The more compact A form of DNA has 11 base pairs per turn, with a much deeper major groove and a much shallower minor groove than B-form DNA. - Important modifications in the structure of standard B-form DNA come about as a result of protein binding to specific DNA sequences. - Although the multitude of hydrogen and hydrophobic bonds between the bases provides stability to DNA, the double helix is flexible about its long axis. - Unlike the a helix in proteins (Figure 3), it has no hydrogen bonds parallel to the axis of the helix. This property allows DNA to bend when complexed with a DNA-binding protein, such as the transcription factor TBP (Figure -4). - Figure (3): The a helix, a common secondary structure in proteins. - The polypeptide backbone (seen as a ribbon) is folded into a spiral that is held in place by hydrogen bonds between backbone oxygen and hydrogen atoms. - Only hydrogens involved in bonding are shown. - The outer surface of the helix is covered by the side-chain R groups (green). - Figure (5):Interaction with a protein can bend DNA. - The conserved C-terminal domain of the TATA box-binding protein (TBP) binds to the minor groove of specific DNA sequences rich in A and T, untwisting and sharply bending the double helix. Transcription of most eukaryotic genes requires participation of TBP. - **Access to Information with the Double Helix without Breaking it Apart:** - How can the information held within DNA sequence of be read without breaking the double helix? - The constant nature of the sugar-phosphate backbone would seem to provide an almost a barrier to ‘reading’ the DNA base sequence. - The grooves along the helical axis provide a mechanism whereby the bases can be distinguished from one another. (DNA is composed of alternating major and minor grooves along its axis). - Bending DNA is also critical to the dense packing of DNA in chromatin, the protein-DNA complex in which nuclear DNA occurs in eukaryotic cells. - Why did DNA, rather than RNA, evolve to be the carrier of genetic information in cells? - The hydrogen at the 2’ position in the deoxyribose of DNA makes it a far more stable molecule than RNA, which instead has a hydroxyl group at the 2’ position of ribose (Figure -6a). - The 2’-hydroxyl groups in RNA participate in the slow, OH--catalyzed hydrolysis of phosphodiester bonds at neutral pH (Figure -6b). - The absence of 2'-hydroxyl groups in DNA prevents this process. - Therefore, the presence of deoxyribose in DNA makes it a more stable molecule—a characteristic that is critical to its function in the long-term storage of genetic information. - Figure (6a and b):Base-catalyzed hydrolysis of RNA. - The 2'-hydroxyl group in RNA can act as a nucleophile, attacking the phosphodiester bond. - The 2’,3’ cyclic monophosphate derivative is further hydrolyzed to a mixture of 2’ and 3’ monophosphates. This mechanism of phosphodiester bond hydrolysis cannot occur in DNA, which lacks 2’-hydroxyl groups. - A structure for Deoxyribose Nucleic Acid - We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.). This structure has novel features which are of considerable biological interest - A structure for nucleic acid has already been proposed by Pauling and Corey. They kindly made their manuscript available to us in advance of publication. - Their model consists of three intertwined chains, with the phosphates near the fibre axis, and the bases on the outside. - In our opinion, this structure is unsatisfactory for two reasons: - We believe that the material which gives the X-ray diagrams is the salt, not the free acid. Without the acidic hydrogen atoms it is not clear what forces would hold the structure together, especially as the negatively charged phosphates near the axis will repel each other. - Some of the van der Wauls distances appear to be too small. - Another three-chain structure has also been suggested by Fraver (in the press). In his model the phosphates are on the outside and the bases are on the inside, linked together by hydrogen bonds. This structure as described is rather ill-defined, and for this reason we shall not comment on it. - We wish to put forward a radically different structure for the salt of deoxyribose nucleic acid. This structure has two helical chains each coiled round the same axis (see diagram). We have made the usual chemical assumptions, namely, (1) that each chain consists of phosphate diester groups joining 3' to 5' deoxyribofuranose residues with 3', 5' linkages. - The two chains (both chains follow right-handed helices, but owing to the dyad the sequences of the atoms in the two chains run in opposite directions. Each chain loosely resembles Furberg's model No. 1: that is the bases are on the inside of the helix and the phosphates on the outside. The configuration of the sugar and the atoms near it is close to Furberg's standard configuration, the sugar being deeply perpendicular to each base. There is a residue on each chain every 3-4 A in the z-direction. We have assumed an angle of 36" between adjacent residues in the same chain, so that the structure repeats after 10 residues on each chain, that is, after 34 A. The distance of a phosphorus atom from the fibre axis is 10 A. As the phosphates are on the outside, cations have easy access to them. - The structure is an open one, and as water content is rather high. At lower water contents we would expect the bases to tilt so that the structure could become more compact. - The novel feature of the structure is the manner in which the two chains are held together by the purine and pyramidine bases The planes of the bases are perpendicular to the fibre axis. They are joined together in pairs, a single base from one chain being hydrogen-bonded to a single base from the other chain, so that the two lie side by side with identical z-coordinates. - One of the pair must be a purine and the other a pyrimidine for bonding to occur. - The hydrogen bonds are made as follows: purine position 1 to pyramidine position 1, purine position 6 to pyramidine position 6. - If it is assumed that the bases only occur in the structure in the most plausible tautomeric forms (that is, with the keto rather than the enol configurations), it is found that only specific pairs of bases can bond together. These pairs are adenine (purine) with thymine (pyrimidine), and guanine (purine) with cytosine (pyrimidine). - In other words, if an adenine forms one member of a pair, on either chain, then on that chain the other member must be thymine, similarly for guanine and cytosine. The sequence of bases on a single chain does not appear to be restricted in any way. - However, if only specific pairs of bases can be formed, it follows that if the sequence of bases on one chain is given, then the sequence on the other chain is automatically determined. - It has been found experimentally that the ratio of the amounts of adenine to thymine, and the ratio of guanine to cytosine, are always very close to unity for deoxyribose nucleic acid. - It is probably impossible to build this structure with a ribose sugar in place of the deoxyribose, as the extra oxygen would make two close a van der Waals contact. - The previously published X-ray data on deoxyribose nucleic acid are insufficient for a rigorous test of our structure. - So far as we can tell, it is roughly compatible with the experimental data, but it must be regarded as unproved until it has been checked against more exact results. Some of these are given in the following communications. We were not aware of the details of the results presented there when we devised our structure, which rests mainly (though not entirely) on published experimental data and theoretical arguments. - It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. - Full details of the structure, including the conditions assumed in building it, together with a set of coordinates for the atoms, will be published elsewhere. - We are much indebted to Dr. Jerry Donohue for constant advice and criticism, especially on interatomic distances. We have also been stimulated by a knowledge of the general nature of the unpublished experimental results and ideas of Dr. M. H. F. Wilkins, Dr. R. E. Franklin and their co-workers at King’s College, London. - One of us (J. D. W.) has been aided by a fellowship from the National Foundation for Infantile Paralysis. - Figure 3.08 DNA Is a Double Helix - This one-page paper published in Nature described the now-famous double helix. J. D. Watson & F. H. C. Crick, Molecular Structure of Nucleic Acids, A Structure for Deoxyribose Nucleic Acid, Nature 171 (1953) 737. - In 2003 the Double Helix celebrated its 50th anniversary. In Great Britain, the Royal Mail issued a set of five commemorative stamps illustrating the double helix together with some of the technological advances that followed, such as comparative genomics and genetic engineering. In addition, the Royal Mint issued a £2 coin depicting the DNA double helix itself (Fig. 3.09). - Figure 3.09 Double Hellix-50th Anniversary Coin - A £2 coin commemorating the discovery of the double helix was issued in 2003 by Great Britain. ## A - DNA, B - DNA, Z - DNA characteristics | characteristic | A - DNA | B - DNA | Z - DNA | |-----------------------------|-----------------------|----------------------|---------------------------------------| | Conditions required to produce | 75% H₂O | 92% H₂O | Alternating purine and pyrimidine bases | | structure | | | | | Helix direction | Right-handed | Right-handed | Left-handed | | Average base pairs per turn | 11 | 10 | 12 | | Rotation per base pair | 32.7° | 36°-30° | | | Distance between adjacent bases | 0.26 nm | 0.34 nm | 0.37 nm | | Diameter | 2.3 nm | 1.9 nm | 1.8 nm | | Overall shape | Short and wide | Long and narrow | Elongated and narrow | - If the meaning of B - DNA wasn't clear to you, it is the common one.