1. Reginald H. Garrett Charles M. Grisham Chapter 10 Nucleotides and Nucleic Acids

2. Chapter 10 “We have discovered the secret of life.” Francis Crick, to patrons of The Eagle, a pub in Cambridge, England (1953) Francis Crick (right) and James Watson (left) point out features of their model for the structure of DNA.

3. Essential Questions What are the structures of the nucleotides? How are nucleotides joined together to form nucleic acids? How is information stored in nucleic acids? What are the biological functions of nucleotides and nucleic acids?

4. Outline What are the structure and chemistry of nitrogenous bases? What are nucleosides? What are the structure and chemistry of nucleotides? What are nucleic acids? What are the different classes of nucleic Acids? Are nucleic acids susceptible to hydrolysis?

5. Information Transfer in Cells Information encoded in a DNA molecule is transcribed via synthesis of an RNA molecule The sequence of the RNA molecule is "read" and is translated into the sequence of amino acids in a protein See Figure 10.1

6. Information Transfer in Cells Figure 10.1 The fundamental process of information transfer in cells.

7. 10.1 What Are the Structure and Chemistry of Nitrogenous Bases? Know the basic structures Pyrimidines Cytosine (DNA, RNA) Uracil (RNA) Thymine (DNA) Purines Adenine (DNA, RNA) Guanine (DNA, RNA)

8. 10.1 What Are the Structure and Chemistry of Nitrogenous Bases? Figure 10.2 (a) The pyrimidine ring system; by convention, atoms are numbered as indicated. (b) The purine ring system; atoms numbered as shown.

9. 10.1 What Are the Structure and Chemistry of Nitrogenous Bases? Figure 10.3 The common pyrimidine bases – cytosine, uracil, and thymine – in the tautomeric forms predominant at pH 7.

10. 10.1 What Are the Structure and Chemistry of Nitrogenous Bases? Figure 10.4 The common purine bases – adenine and guanine – in the tautomeric forms predominant at pH 7.

11. 10.1 What Are the Structure and Chemistry of Nitrogenous Bases? Figure 10.5 Other naturally occurring purine derivatives – hypoxanthine, xanthine, and uric acid.

12. The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature The aromaticity and electron-rich nature of pyrimidines and purines enable them to undergo keto-enol tautomerism The keto tautomers of uracil, thymine, and guanine predominate at pH 7 By contrast, the enol form of cytosine predominates at pH 7 Protonation states of the nitrogens determines whether they can serve as H-bond donors or acceptors Aromaticity also accounts for strong absorption of UV light

13. The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature Figure 10.6 The keto-enol tautomerism of uracil.

14. The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature Figure 10.7 The tautomerization of the purine guanine.

15. The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature Figure 10.8 The UV absorption spectra of the common ribonucleotides.

16. The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature Figure 10.8 The UV absorption spectra of the common ribonucleotides.

17. Adenosine: A Nucleoside with Physiological Activity Adenosine functions as an autacoid, or local hormone, and neuromodulator. Circulating in the bloodstream, it influences blood vessel dilation, smooth muscle contraction, neurotransmitter release, and fat metabolism. Adenosine is also a sleep regulator. Adenosine rises during wakefulness, promoting eventual sleepiness. Caffeine promotes wakefulness by blocking binding of adenosine to its neuronal receptors.

18. 10.2 What Are Nucleosides? Structures to Know Nucleosides are compounds formed when a base is linked to a sugar via a glycosidic bond The sugars are pentoses D-ribose (in RNA) 2-deoxy-D-ribose (in DNA) The difference - 2'-OH vs 2'-H This difference affects secondary structure and stability

19. 10.2 What Are Nucleosides? Figure 10.9 The linear and cyclic (furanose) forms of ribose.

20. 10.2 What Are Nucleosides? Figure 10.9 The linear and cyclic (furanose) forms of deoxyribose.

21. 10.2 What Are Nucleosides? The base is linked to the sugar via a glycosidic bond The carbon of the glycosidic bond is anomeric Named by adding -idine to the root name of a pyrimidine or -osine to the root name of a purine Conformation can be syn or anti Sugars make nucleosides more water-soluble than free bases

22. 10.2 What Are Nucleosides? Figure 10.10 The common ribonucleosides.

23. 10.3 What Is the Structure and Chemistry of Nucleotides? Nucleotides are nucleoside phosphates Know the nomenclature "Nucleotide phosphate" is redundant! Most nucleotides are ribonucleotides Nucleotides are polyprotic acids

24. 10.3 What Is the Structure and Chemistry of Nucleotides? Figure 10.11 Structures of the four common ribonucleotides – AMP, GMP, CMP, and UMP. Also shown: 3’-AMP.

25. 10.3 What Is the Structure and Chemistry of Nucleotides? Figure 10.12 The cyclic nucleotide cAMP.

26. 10.3 What Is the Structure and Chemistry of Nucleotides? Figure 10.12 The cyclic nucleotide cGMP

27. 10.3 What Is the Structure and Chemistry of Nucleotides? Figure 10.13 Formation of ADP and ATP by the succesive addition of phosphate groups via phosphoric anhydride linkages. Note that the reaction is a dehydration synthesis.

28. 10.3 What Is the Structure and Chemistry of Nucleotides? Figure 10.13 Formation of ADP and ATP by the succesive addition of phosphate groups via phosphoric anhydride linkages. Note that the reaction is a dehydration synthesis.

29. Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy Nucleoside 5’-triphosphates are indispensable agents in metabolism because their phosphoric anhydride bonds are a source of chemical energy Bases serve as recognition units Cyclic nucleotides are signal molecules and regulators of cellular metabolism and reproduction ATP is central to energy metabolism GTP drives protein synthesis CTP drives lipid synthesis UTP drives carbohydrate metabolism

30. Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy Figure 10.14 Phosphoryl, pyrophosphoryl, and nucleotidyl group transfer, the major biochemical reactions of nucleotides. Phosphoryl group transfer is shown here.

31. Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy Figure 10.14 Phosphoryl, pyrophosphoryl, and nucleotidyl group transfer, the major biochemical reactions of nucleotides. Pyrophosphoryl group transfer is shown here.

32. Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy Figure 10.14 Phosphoryl, pyrophosphoryl, and nucleotidyl group transfer, the major biochemical reactions of nucleotides. Nucleotidyl group transfer is shown here.

33. 10.4 What Are Nucleic Acids? Nucleic acids are linear polymers of nucleotides linked 3' to 5' by phosphodiester bridges Ribonucleic acid and deoxyribonucleic acid Know the shorthand notations Sequence is always read 5' to 3' In terms of genetic information, this corresponds to "N to C" in proteins

34. 10.4 What Are Nucleic Acids? Figure 10.15 3',5'- phosphodiester bridges link nucleotides together to form polynucleotide chains. The 5'- ends of the chains are at the top; the 3'-ends are at the bottom.

35. 10.4 What Are Nucleic Acids? Figure 10.15 3’,5’- phosphodiester bridges link nucleotides together to form polynucleotide chains. The 5’-ends of the chains are at the top; the 3’-ends are at the bottom.

36. 10.5 What Are the Different Classes of Nucleic Acids? DNA - one type, one purpose RNA - 3 (or 4) types, 3 (or 4) purposes ribosomal RNA - the basis of structure and function of ribosomes messenger RNA - carries the message for protein synthesis transfer RNA - carries the amino acids for protein synthesis Others: Small nuclear RNA Small non-coding RNAs

37. 10.5 What Are the Different Classes of Nucleic Acids? Figure 10.16 The antiparallel nature of the DNA double helix.

38. The DNA Double Helix The double helix is stabilized by hydrogen bonds "Base pairs" arise from hydrogen bonds Erwin Chargaff had the pairing data, but didn't understand its implications Rosalind Franklin's X-ray fiber diffraction data was crucial Francis Crick showed that it was a helix James Watson figured out the H bonds

39. Chargaff’s Data Held the Clue to Base Pairing

40. http://www.youtube.com/watch?v=IRNE_IfFNZE

41. The Base Pairs Postulated by Watson Figure 10.17 The Watson-Crick base pairs A:T and G:C.

42. The Base Pairs Postulated by Watson Figure 10.17 The Watson-Crick base pairs A:T and G:C.

43. The Structure of DNA An antiparallel double helix Diameter of 2 nm Length of 1.6 million nm (E. coli) Compact and folded (E. coli cell is only 2000 nm long) Eukaryotic DNA wrapped around histone proteins to form nucleosomes Base pairs: A-T, G-C

44. The Structure of DNA Figure 10.18 Replication of DNA gives identical progeny molecules because base pairing is the mechanism that determines the nucleotide sequence of each newly synthesized strand.

45. Digestion of the E. coli cell wall releases the bacterial chromosome Figure 10.19 The chromosome is shown surrounding the cell.

46. Do the Properties of DNA Invite Practical Applications? The molecular recognition between DNA strands can create a molecule with mechanical properties different from single-stranded DNA DNA double helices are relatively rigid rods DNA chains have been used to construct nanomachines capable of simple movements such as rotation and pincerlike motions More elaborate DNA-based devices can act as motors walking along DNA tracks The construction of “DNA tweezers” is described on the following slide

48. Do the Properties of DNA Invite Practical Applications? DNA tweezers – a simple DNA nanomachine.

49. Messenger RNA Carries the Sequence Information for Synthesis of a Protein Transcription product of DNA In prokaryotes, a single mRNA contains the information for synthesis of many proteins In eukaryotes, a single mRNA codes for just one protein, but structure is composed of introns and exons

50. Messenger RNA Carries the Sequence Information for Synthesis of a Protein Figure 10.20 Transcription and translation of mRNA molecules in prokaryotic versus eukaryotic cells. In prokaryotes, a single mRNA molecule may contain the information for the synthesis of several polypeptide chains within its nucleotide sequence.

51. Messenger RNA Carries the Sequence Information for Synthesis of a Protein Figure 10.20 Transcription and translation of mRNA molecules in prokaryotic versus eukaryotic cells. Eukaryotic mRNAs encode only one polypeptide but are more complex.

52. Eukaryotic mRNA DNA is transcribed to produce heterogeneous nuclear RNA mixed introns and exons with poly A intron - intervening sequence exon - coding sequence poly A tail - stability? Splicing produces final mRNA without introns

53. Ribosomal RNA Provides the Structural and Functional Foundation for Ribosomes Ribosomes are about 2/3 RNA, 1/3 protein rRNA serves as a scaffold for ribosomal proteins The different species of rRNA are referred to according to their sedimentation coefficients rRNAs typically contain certain modified nucleotides, including pseudouridine and ribothymidylic acid The role of ribosomes in biosynthesis of proteins is treated in detail in Chapter 30 Briefly: the genetic information in the nucleotide sequence of mRNA is translated into the amino acid sequence of a polypeptide chain by ribosomes

54. Ribosomal RNA Provides the Structural and Functional Foundation for Ribosomes Figure 10.21 Ribosomal RNA has a complex secondary structure due to many intrastrand H bonds. The gray line here traces a polynucleotide chain consisting of more than 1000 nucleotides. Aligned regions represent H- bonded complementary base sequences.

55. Ribosomal RNA Provides the Structural and Functional Foundation for Ribosomes Figure 10.22 The organization and composition of ribosomes.

56. Ribosomal RNA Provides the Structural and Functional Foundation for Ribosomes Figure 1-23 Unusual bases in DNA.

57. Ribosomal RNA Provides the Structural and Functional Foundation for Ribosomes Figure 10.23 Unusual bases in DNA.

58. Transfer RNAs Carry Amino Acids to Ribosomes for Use in Protein Synthesis Small polynucleotide chains - 73 to 94 residues each Several bases usually methylated Each a.a. has at least one unique tRNA which carries the a.a. to the ribosome 3'-terminal sequence is always CCA-a.a. Aminoacyl tRNA molecules are the substrates of protein synthesis

59. Transfer RNAs Carry Amino Acids to Ribosomes for Use in Protein Synthesis Figure 10.24 Transfer RNA also has a complex secondary structure due to many intrastrand hydrogen bonds. The black lines represent base-paired nucleotides in the sequence.

60. The RNA World and Early Evolution Thomas Cech and Sidney Altman showed that RNA molecules are not only informational – they can also be catalytic This gave evidence to the postulate by Francis Crick and others that prebiotic evolution depended on self- replicating, catalytic RNAs But what was the origin of the nucleotides? A likely source may have been conversion of aminoimidazolecarbonitrile to adenine And glycolaldehyde could combine with other molecules to form ribose Adenine and glycolaldehyde exist in outer space

61. The RNA World and Early Evolution Aminoimidazolecarbonitrile is a pentamer of HCN and may be a celestial precursor of adenine.

62. The RNA World and Early Evolution Glycolaldehyde has been detected at the center of the Milky Way and could be a precursor of ribose and glucose.

63. The Chemical Differences Between DNA and RNA Have Biological Significance Two fundamental chemical differences distinguish DNA from RNA: DNA contains 2-deoxyribose instead of ribose DNA contains thymine instead of uracil

64. The Chemical Differences Between DNA and RNA Have Biological Significance Why does DNA contain thymine? Cytosine spontaneously deaminates to form uracil Repair enzymes recognize these "mutations" and replace these Us with Cs But how would the repair enzymes distinguish natural U from mutant U Nature solves this dilemma by using thymine (5-methyl-U) in place of uracil

65. The Chemical Differences Between DNA and RNA Have Biological Significance Figure 10.25 Deamination of cytosine forms uracil.

66. The Chemical Differences Between DNA and RNA Have Biological Significance Figure 10.26 The 5-methyl group on thymine labels it as a special kind of uracil.

67. DNA & RNA Differences? Why is DNA 2'-deoxy and RNA is not? Vicinal -OH groups (2' and 3') in RNA make it more susceptible to hydrolysis DNA, lacking 2'-OH is more stable This makes sense - the genetic material must be more stable RNA is designed to be used and then broken down

68. 10.6 Are Nucleic Acids Susceptible to Hydrolysis? RNA is resistant to dilute acid DNA is depurinated by dilute acid DNA is not susceptible to base RNA is hydrolyzed by dilute base See Figure 10.29 for mechanism

69. 10.6 Are Nucleic Acids Susceptible to Hydrolysis? Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilic attach by OH - on the P atom leads to 5'-phosphoester cleavage.

70. 10.6 Are Nucleic Acids Susceptible to Hydrolysis? Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilic attach by OH - on the P atom leads to 5'-phosphoester cleavage. Random hydrolysis of the cyclic phosphodiester intermediate gives a mixture of 2'- and 3'-nucleoside monophosphate products.

71. 10.6 Are Nucleic Acids Susceptible to Hydrolysis? Figure 10.27 Alkaline hydrolysis of RNA. Random hydrolysis of the cyclic phosphodiester intermediate gives a mixture of 2'- and 3'-nucleoside monophosphate products.

72. 10.6 Are Nucleic Acids Susceptible to Hydrolysis? Figure 10.28 Cleavage in polynucleotide chains. Cleavage on the a side leaves the phosphate attached to the 5'- position of the adjacent nucleotide. b-side hydrolysis yields 3'-phosphate products.

73. 10.6 Are Nucleic Acids Susceptible to Hydrolysis? Figure 10.28 Cleavage in polynucleotide chains. Cleavage on the a side leaves the phosphate attached to the 5'-position of the adjacent nucleotide.

74. 10.6 Are Nucleic Acids Susceptible to Hydrolysis? Figure 10.28 Cleavage in polynucleotide chains. b-side hydrolysis yields 3'-phosphate products.

75. Restriction Enzymes Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes" Type II and III restriction enzymes cleave DNA chains at selected sites Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage An enzyme that recognizes a 6-base sequence is a "six-cutter"

76. Type II Restriction Enzymes No ATP requirement Recognition sites in dsDNA have a 2-fold axis of symmetry Cleavage can leave staggered or "sticky" ends or can produce "blunt” ends

77. Type II Restriction Enzymes Names use 3-letter italicized code: 1st letter - genus; 2nd,3rd - species Following letter denotes strain EcoRI is the first restriction enzyme found in the R strain of E. coli

78. Cleavage Sequences of Restriction Endonucleases

81. Restriction Mapping of DNA Figure 10.29 Restriction mapping analysis.