Chapter 2: The Structure of DNA

1. Chapter 2: The Structure of DNA

2. Imagine yourself inside one of those cooling DNA solutions, observing the rebirth of beautifully undulating, semirigid, double-helical threads from the jumble of billions of intertwisted single strands. It is a mind reeling spectacle. Christian de Duve, A Guided Tour of the Living Cell (1984), p. 292

3. 2.1 Introduction

4. Objectives • Describe the structure of DNA • Keep its function within living cells in mind

5. 2.2Primary structure: the components of nucleic acids

6. Components of nucleotides • Five-carbon sugars • Nitrogenous bases • The phosphate functional group

7. Sugars • The carbons of the sugar are given numbers from 1’ to 5’. • The sugar of DNA is deoxyribose, which lacks 2’-OH. • The sugar of RNA is ribose. 5’ 4’ 1’ 2’ 3’ lacks 2’-OH

9. Pyrimidines • The atoms of the pyrimidines are given numbers from 1 to 6.

10. Purines • The atoms of purines are given numbers from 1 to 9.

11. Edwin Chargaff’s “rules” [A] = [T] [G] = [C] [A] + [G] = [T] + [C] • %G+C differs among species but is constant in all cells of an organism within a species. • Varies from 22 to 73%

12. Nucleosides and nucleotides DNA and RNA chains are formed through a series of three steps: • A base attached to a sugar is a nucleoside. • A nucleoside with one or more phosphates attached is a nucleotide. • Nucleotides are linked by 5′ to 3′ phosphodiester bonds between adjacent nucleotides to form a DNAorRNA chain.

13. Structure of nucleosides: base + sugar • Nucleosides are derivatives of purines and pyrimidines that have sugar linked to a ring nitrogen: N9 for purines N1 for pyrimidines via a β-N-glycosidic bond. β-N-glycosidic bond

14. Structure of nucleotides • Nucleotides are composed of nucleosides (base + sugar) and 1, 2, or 3 groups of phosphate. • Mononucleotide (nucleoside-monophosphate)= nucleoside + 1 phosphate • Nucleoside-diphosphate = nucleoside + 2 phosphates • Nucleoside-triphosphate = nucleoside + 3 phosphates

15. The components of a DNA or RNA chain are joined by covalent bonds. • A covalent bond is a strong chemical bond formed when electrons are shared between two atoms. • These bonds are very stable and do not break spontaneously within cells.

16. Nomenclature of nucleotides Example: the base cytosine (C) DNA deoxycytidine 5′-triphosphate (dCTP) RNA cytidine 5′-triphosphate (CTP) Generic deoxynucleoside 5′-triphosphate (dNTP) nucleoside 5′-triphosphate (NTP)

17. Physiological functions of nucleotides • “Bricks” for nucleic acids synthesis • Macroergic compounds which deliver energy necessary to different biological processes (eg. ATP, GTP) • Allosteric regulators of different enzymes • Methyl group donor (eg. S-adenosylmethionine) • Signal transduction: intracellular messengers of hormones (eg. AMPc, GMCc)

18. Free natural nucleotides: cAMP • 3’,5’cyclic adenosine monophosphate (cAMP) serves as a second messenger or transducer of information between the extracellular and intracellular medium.

19. The length of RNA and DNA • RNA The number of nucleotides (nt) or bases is used as a measure of length. • Double-stranded DNA The number of base pairs (bp) is used as a measure of length. 1000 bp = 1 kilobase pair (kb or kbp) 1,000,000 = 1 megabase pair (Mb or Mbp)

20. Natural RNAs come in sizes ranging from less than one hundred to many thousands of nucleotides • DNA can be as long as several kb to thousands of Mb • Oligonucleotides are short chains of single-stranded DNA (< 50 bases)

21. Significance of 5′ and 3′ • The 5′-PO4 and 3′-OH ends of a DNA or RNA chain are distinct and have different properties • By convention, a DNA sequence is written with the 5′ end to the left and the 3′ end to the right

22. Polynucletide chain of DNA(primary/covalent structure) 1’ • The polynucleotide chain of DNA consist of nucleotides joined together by 3’,5’ phosphodiester bonds. • Phosphodiester bonds link the 3’- and 5’-sugar carbons of adiacent monomer. • Polynucleotides are directional macromolecules: each end of a polymer is distinct. • 3’ –end is one with a free 3’-hydroxyl. • 5’ –end is one with a free or phosphorilated 5’-hydroxyl. • Polynucleotides bear a negative charge at physiological pH 3’linkage 5’linkage

23. 2.3Secondary structure of DNA

24. What chemical forces hold (or drive) the DNA strands together? (also applies to double-stranded regions of RNA)

25. 1. Hydrogen bonds between bases Also important that the purine-pyrimidine base pairs are of similar size.

26. Hydrogen bonds form between the bases • Two common “Watson-Crick” or “complementary” base pairs: Adenine (A) is joined to thymine (T) by two hydrogen bonds. Guanine (G) is joined to cytosine (C) by three hydrogen bonds.

27. Why aren’t there other stable base pairs present in DNA? • May not be able to form two or more hydrogen bonds. • Pairing of G with T produces a pair with a similar shape to Watson-Crick base pairs. • Fidelity of DNA replication: proofreading and DNA repair mechanisms correct mistakes. • GU base-pairing is of importance in RNA structure.

28. Base stacking provides chemical stability to the DNA double helix • The hydrophobic nitrogenous bases stack onto each other without a gap by means of a helical twist. • A double-stranded DNA molecule has a hydrophobic core composed of stacked bases.

29. Hydrophobic bonding is an example of weak van der Waals interactions. • A large number of weak van der Waals interactions can significantly increase the stability of a structure, such as the DNA double helix.

30. Structure of the Watson-Crick DNA double helix • Polarity in each strand: 5′ 3′ • Two strands are antiparallel • Major and minor grooves

32. 3 Ways of Depicting DNA Structure

33. Major and minor grooves • The major groove carries a “message” that can be read by DNA binding proteins.. • In the major groove, the pattern of hydrogen-bonding groups is different for AT, TA, GC, and CG base pairs. • In the minor groove, there is only one difference in the pattern between AT and GC base pairs.

34. Distinguishing between features of alternative double-helical structures • B-DNA (Watson-Crick DNA) • A-DNA • Z-DNA

37. Higher Order RNA Structure Stem-loops are common elements of secondary RNA structure. Stems are double-stranded regions of RNA that are A-form helices. They usually follow Watson-Crick base pairing rules (U replaces T), but other pairs occur (G – U is common). (DNA is typically a B-form helix). Stem loop

38. The predominant form of DNA in vivo is B-DNA. • But, there is evidence for a role of Z-DNA in vivo: • Z-DNA binding proteins. • Short sections of Z-DNA within a cell are energetically favorable and stable. • Role in regulating gene expression?

39. A region of Z-DNA is connected to B-DNA through a junction in which one base pair is flipped out, or extruded, from the DNA helix. • This process is called base flipping.

40. DNA can undergo reversible strand separation Significance of complementary base pairing: • Fidelity of DNA replication, transcription, and translation. • Ability to manipulate in the lab by denaturation, renaturation, and hybridization.

41. Denaturation or “melting” of DNA • Base stacking in duplex DNA quenches the capacity of bases to absorb UV light. • Hyperchromicity: As DNA “melts” its absorption of UV light increases. • Tm(melting temperature): The temperature at which half of the bases in a dsDNA sample have denatured.

42. Renaturation or “reannealing”of DNA • The capacity to renature denatured DNA permits hybridization. • Hybridization is the complementary base pairing of strands from two different sources. • The rate at which DNA reanneals is a function of the length of the DNA and the initial concentration in the sample.

43. A DNA renaturation Cot curve • C/C0 = 1/[1 + KC0t] • The expression C0t is called “Cot.” • Cot ½ is when renaturation is half completed. • A plot of C/C0 versus C0t is called a Cot curve.

44. Comparison of Cot curves for E. coli and calf thymus DNA • The Cot ½ of calf thymus DNA is greater than the Cot ½ of E. coli DNA. • Explanation: The larger the genome size, the longer it will take for any one sequence to encounter its complementary sequence.

45. Denaturation and Reannealing

46. DNA Denaturation Curve

47. Dependence of DNA denaturation on G + C content andon salt concentration

48. The E. coli DNA curve resembles an ideal Cot curve. • The calf thymus DNA curve is not smooth. • Explanation: the rapidly reannealing fraction in the calf DNA represents repeated DNA sequences; the more slowing reannealing fraction represents unique sequences.

49. 2.4Unusual DNA secondary structures

50. Slipped structures • Occur at tandem repeats • Found upstream of regulatory regions