1. Nucleotides and Nucleic Acids
Chapter 19
Nucleotides and Nucleic Acids 1

2. 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. 3

3. Know the basic structures
Nitrogenous Bases
Know the basic structures
Pyrimidines
Cytosine (DNA, RNA)
Uracil (RNA)
Thymine (DNA)
Purines
Adenine (DNA, RNA)
Guanine (DNA, RNA)
Cytosine (C)
Thymine (T)
Uracil (U)
DNA & RNA
DNA
RNA
Adenine (A)
Guanine (G)
DNA & RNA 4

4. Properties of Pyrimidines and Purines
Keto-enol tautomerism 5

5. Properties of Pyrimidines and Purines
Acid/base dissociations
Strong absorbance of UV light 5

6. Pentoses of Nucleotides
Know these structures too
D-ribose (in RNA)
2-deoxy-D-ribose (in DNA)
The difference - 2'-OH vs 2'-H
This difference affects secondary structure and stability 6

7. Linkage of a base to a sugar
11.3 Nucleosides
Linkage of a base to a sugar
Base is linked via a glycosidic bond
The carbon of the glycosidic bond is anomeric 7

8. Linkage of a base to a sugar
11.3 Nucleosides
Linkage of a base to a sugar
Named by adding -idine to the root name of a pyrimidine or -osine to the root name of a purine 7

9. Linkage of a base to a sugar
11.3 Nucleosides
Linkage of a base to a sugar
Conformation can be syn or anti
Sugars make nucleosides more water-soluble than free bases 7

10. Nucleoside phosphates
11.4 Nucleotides
Nucleoside phosphates
Nucleotides are nucleosides esterified with phosphoric acid
Most are esterified at the 3’ or 5’ position
"Nucleotide phosphate" is redundant!
Nucleotides are poly-protic acids 8

11. Nucleoside phosphates
11.4 Nucleotides
Nucleoside phosphates 8

12. Functions of Nucleotides
Nucleoside 5'-triphosphates are carriers of energy
Bases serve as recognition units 9

13. Functions of Nucleotides
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 9

14. 11.5 Nucleic Acids - Polynucleotides
Following are names and one-letter abbreviations for the heterocyclic aromatic amine bases most common to nucleic acids 10

15. 11.5 Nucleic Acids - Polynucleotides
Polymers linked 3' to 5' by phosphodiester bridges
Ribonucleic acid (RNA) and deoxyribonucleic acid(DNA)
Know the shorthand notations
Sequence is always read 5' to 3'
In terms of genetic information, this corresponds to "N to C" in proteins
T-G is a dinucleotide 10

16. 11.5 Nucleic Acids - Polynucleotides10

17. 11.5 Nucleic Acids - Polynucleotides10

18. 11.6 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
transfer RNA - carries the amino acids 11

19. Structure of DNA
Primary Structure: the sequence of bases along the pentose-phosphodiester backbone of a DNA molecule (or an RNA molecule) read from the 5’ end to the 3’ end
Secondary structure: the ordered arrangement of nucleic acid strands
Double helix: a type of 2° structure of DNA molecules in which two antiparallel polynucleotide strands are coiled in a right-handed manner about the same axis

20. Stabilized by hydrogen bonds!
The DNA Double Helix
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 knew it was a helix
James Watson figured out the H-bonds 12

21. DNA - 2° Structure
T-A base pairing

22. DNA - 2° Structure
C-G base pairing

23. DNA - 2° Structure
Ribbon model of B-DNA

26. DNA - 2° Structure
B-DNA
the predominant form in dilute aqueous solution
a right-handed helix
20 Å thick with 34 Å per ten base pairs
minor groove of 12 Å and major groove of 22 Å
A-DNA
a right-handed helix, but thicker than B-DNA
29 Å per 10 base pairs
Z-DNA
a left-handed double helix

27. The Structure of DNA 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 13

28. DNA - 3° Structure
Tertiary structure: the three-dimensional arrangement of all atoms of a nucleic acid, commonly referred as to supercoiling
Circular DNA: a type of double-stranded DNA in which the 5’ and 3’ ends of each stand are joined by a phosphodiester bond (Fig 20.10)
Chromatin: consists of DNA molecules wound around particles of histones in a beadlike structure

29. DNA - 3° Structure
Relaxed, strained, and supercoiled DNA

30. Ribonucleic Acids (RNA)
RNA are similar to DNA in that they, too, consist of long, unbranched chains of nucleotides joined by phosphodiester bonds between the 3’-OH of one pentose and the 5’-OH of the next; however:
the pentose unit in RNA is -D-ribose rather than -2-deoxy-D-ribose
the pyrimidine bases in RNA are uracil and cytosine rather than thymine and cytosine
RNA is single stranded rather than double stranded

31. RNA
RNA molecules are classified according to their structure and function
Ribosomal RNA (rRNA): a ribonucleic acid found in ribosomes, the site of protein synthesis

32. RNA
Transfer RNA (tRNA): a ribonucleic acid that carries a specific amino acid to the site of protein synthesis on ribosomes

33. RNA
Messenger RNA (mRNA): a ribonucleic acid that carries coded genetic information from DNA to the ribosomes for the synthesis of proteins
present in cells in relatively small amounts and very short-lived
single stranded
their synthesis is directed by information encoded on DNA
a complementary strand of mRNA is synthesized along one strand of an unwound DNA, starting from the 3’ end

34. RNA
the synthesis of mRNA from DNA is called transcription

35. Genetic Code

36. Genetic Code Properties of the Code
only 61 triplets code for amino acids; the remaining 3 (UAA, UAG, and UGA) signal chain termination
the code is degenerate, which means that several amino acids are coded for by more than one triplet; Leu, Ser, and Arg, for example, are each coded for by six triplets
for the 15 amino acids coded for by 2, 3, or 4 triplets, it is only the third letter of the codon that varies; Gly, for example, is coded for by GGA, GGG, GGC, and GGU
there is no ambiguity in the code; each triplet codes for one and only one amino acid

37. Sequencing DNA
Restriction endonuclease: an enzyme that catalyzes hydrolysis of a particular phosphodiester bond within a DNA strand
over 1000 endonucleases have been isolated and their specificities determined
typically they recognize a set sequence of nucleotides and cleave the DNA at or near that particular sequence
EcoRI from E. coli, for example, cleaves as shown

38. Sequencing DNA
examples of restriction endonucleases

39. Sequencing DNA
Polyacrylamide gel electrophoresis: a technique so sensitive that it is possible to separate nucleic acid fragments differing from one another in only a single nucleotide
Chain termination or dideoxy method: a method developed by Frederick Sanger for sequencing DNA molecules

40. DNA Replication
the sequence of nucleotides on one strand is copied as a complementary strand to form the second strand of double-stranded DNA
this synthesis is catalyzed by the enzyme DNA polymerase
DNA polymerase will carry out this synthesis in vitro using single-stranded DNA as a template, provided the four dNTPs and a primer are present
because the new DNA strand grows from the 5’ to 3’ end, the primer must have a free 3’-OH group to which the first nucleotide of the growing chain is added

41. Chain-Termination Sequencing
the key is addition of a 2’,3’-dideoxynucleoside triphosphate (ddNTP) to the synthesizing medium
synthesis terminates at any point where a ddNTP becomes incorporated

42. Chain-Termination Sequencing
a single-stranded DNA of unknown sequence is mixed with primer and divided into four separate reaction mixtures
to each mixture is added all four dNTPs, one of which is labeled in its 5’- phosphoryl group with P-32
also added are DNA polymerase and one of the four ddNTPs
when polyacrylamide gel electrophoresis of each reaction mixture is completed, a piece of x-ray film is placed over the gel to detect gamma radiation from the decay of P-32
the base sequence of the complement to the original single-stranded template is read directly from the bottom to top of the developed film

45. Transcription product of DNA
Messenger RNA
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 14

47. 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 15

48. Ribosomal RNA Ribosomes are about 2/3 RNA, 1/3 protein
rRNA serves as a scaffold for ribosomal proteins
23S rRNA in E. coli is the peptidyl transferase! 16

51. Transfer RNA 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 17

52. Why does DNA contain thymine?
DNA & RNA Differences?
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 18

55. Why is DNA 2'-deoxy and RNA is not?
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 19

56. Hydrolysis of Nucleic Acids
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 for mechanism 20

62. 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" 21

63. Type II Restriction Enzymes
No ATP requirement
Recognition sites in dsDNA usually have a 2-fold axis of symmetry
Cleavage can leave staggered or "sticky" ends or can produce "blunt” ends 22

64. 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 23

66. Nucleic Acids
Replication of DNA
NEED ARTWORK FROM BRUICE BOOK

67. Nucleic Acids Enzymes break hydrogen bonds between DNA strands
Replication of DNA T … A A … T C … G C … G A … T G … C A … T … C G C … G T … A
NEED ARTWORK FROM BRUICE BOOK
Enzymes break hydrogen bonds between DNA strands

68. Nucleic Acids
Replication of DNA
Parent strand
Parent strand T A C G … C G A T
NEED ARTWORK FROM BRUICE BOOK
New complementary nucleotides are enzymatically introduced to the separating strands

69. Nucleic Acids Two new complementary daughter strands are produced
Replication of DNA
Parent strand
Parent strand T … A A … T C … G C … G A … T G C A T C G G C C G G C
NEED ARTWORK FROM BRUICE BOOK T A A T
Daughter strands
Two new complementary daughter strands are produced

70. Nucleic Acids Replication is complete, two identical DNA molecules
Replication of DNA
Parent strands … T A C G … T A C G
NEED ARTWORK FROM BRUICE BOOK
Daughter strands
Replication is complete, two identical DNA molecules

71. Nucleic Acids
RNA
Messenger RNA (mRNA) – Carries genetic information from DNA to ribosomes.
Ribosomal RNA (rRNA) – Make up the ribosomes where protein synthesis occurs.
Transfer RNA (tRNA) – Transports amino acids to the ribosomes to make protein.
NEED SOMETHING HERE

72. Nucleic Acids Transcription DNA Coding Strand 5′ 3′ 3′ 5′
DNA Template Strand
NEED SOMETHING HERE

73. Nucleic Acids
Transcription
DNA Coding Strand 5′ 3′ 3′ 5′
DNA Template Strand
NEED SOMETHING HERE
Enzymes open up hydrogen bonds between DNA coding and template strands

74. Nucleic Acids mRNA is synthesized using DNA template Transcription
DNA Coding Strand 5′ 3′ 3′ 5′
DNA Template Strand
NEED SOMETHING HERE 5′ 3′
New mRNA Strand
mRNA is synthesized using DNA template

75. Nucleic Acids Protein Synthesis – Translation
Ribosomes are the site of translation, they are made up of rRNA and protein
mRNA goes to ribosome
Ribonucleotide sequences, codons, on the mRNA tell the ribosome what amino acids are needed in the new protein
NEED SOMETHING HERE
mRNA Strand
Codons

76. Nucleic Acids Arg Ile Ala Phe Protein Synthesis – Translation
Ribosome reads message from codon
NEED SOMETHING HERE
Arg
Ile
Ala
Phe
mRNA Strand
Codons

77. Nucleic Acids Ile U–A–U
Protein Synthesis – Translation
tRNAs bring amino acids
Amino acid is attached to the 3′ end of the tRNA
Ile
U–A–U
NEED SOMETHING HERE
Anticodon

78. Nucleic Acids Polymerase Chain Reaction (PCR)
PCR is used to obtain large quantities of a known sequence of DNA
In just a few hours PCR can amplify 105 molecules of DNA to 1011 molecules
The key enzyme in PCR, Taq, is isolated from the thermophilic bacterium Thermus aquaticus (found in a hot spring in Yellowstone National Park)
NEED SOMETHING HERE

79. Nucleic Acids Polymerase Chain Reaction (PCR) Target DNA
Heat Denatures DNA, separating the strands
95°C
NEED SOMETHING HERE

80. Primers are added, Taq needs double-stranded DNA to begin
Nucleic Acids
Polymerase Chain Reaction (PCR)
Primers are added, Taq needs double-stranded DNA to begin
NEED SOMETHING HERE

81. Nucleic Acids Polymerase Chain Reaction (PCR)
Primers are added, Taq needs double-stranded DNA to begin
Primers are annealed at 50°C
NEED SOMETHING HERE

82. Taq polymerase Mg2+ and nucleotides
Nucleic Acids
Polymerase Chain Reaction (PCR)
Taq polymerase Mg2+ and nucleotides
NEED SOMETHING HERE

83. Nucleic Acids Polymerase Chain Reaction (PCR) Repeat Cycle
NEED SOMETHING HERE
Repeat Cycle
Repeat Cycle