1. Chapter 10 Nucleotides and Nucleic Acids

2. 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 ?

3. Information Transfer in Cells
Central Dogma of Molecular Biology
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.

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

5. 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. N1 is attached to ribose.
(b) The purine ring system; atoms numbered as shown.
N9 is attached to ribose.

6. 10.1 What Are the Structure and Chemistry of Nitrogenous Bases?
Know these structures
Figure The common pyrimidine bases – cytosine, uracil, and thymine – in the tautomeric forms predominant at pH 7.

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

8. 10.1 What Are the Structure and Chemistry of Nitrogenous Bases?
Figure 10.5
Other naturally occurring purine derivatives – hypoxanthine, xanthine, and uric acid.

9. 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 at 260 nm. (Proteins absorb at 280 nm.)

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

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

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

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

14. 10.2 What Are Nucleosides? Structures to Know
Nucleosides are formed when a base is linked to a sugar via a beta glycosidic bond.
The sugars are pentoses.
D-ribose (in RNA).
2-deoxy-D-ribose (in DNA).
The difference - 2'-OH vs 2'-H. Primes are used in nucleosides and nucleotides but not sugars alone.
This difference affects secondary structure and stability.

15. 10.2 What Are Nucleosides?
Figure The linear (Fischer) and cyclic (furanose) forms of ribose.
Figure The linear (Fischer) and cyclic (furanose) forms of deoxyribose.

16. 10.2 What Are Nucleosides?
The base is linked to the sugar via a beta 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. (Uracil  uridine and adenine  adenosine)
Conformation can be syn or anti.
Sugars make nucleosides more water-soluble than free bases.

17. 10.2 What Are Nucleosides?
Figure The common ribonucleosides.

18. 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 due to the phosphates.

19. 10.3 What Is the Structure and Chemistry of Nucleotides?
Figure Structures of the four common ribonucleotides – AMP, GMP, CMP, and UMP. Also shown: 3’-AMP.

20. 10.3 What Is the Structure and Chemistry of Nucleotides?
Figure Formation of ADP from AMP by the addition of a phosphate group forming a phosphoric anhydride linkage. Note that the reaction is a dehydration synthesis.

21. 10.3 What Is the Structure and Chemistry of Nucleotides?
Figure Formation of ATP from ADP by the addition of a phosphate group forming a phosphoric anhydride linkage. This is also a dehydration process.

22. 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.
Functions:
1. ATP is central to energy metabolism: (see the following slides).
2. Nucleotides serve as signal molecules and regulators: c-AMP and c-GMP.
3. NTPs are substrates for DNA and RNA synthesis. The bases serve as recognition units.

23. Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy
Functions continued:
4. Nucleotides are high energy carrier molecules:
GTP is involved in protein synthesis (translation).
Initiation, elongation and termination steps.
And ATP in activation: Aminoacyl-AMP.
CTP is involved in lipid synthesis.
CDP-diacylglycerol, etc.
UTP is involved in carbohydrate metabolism.
UDP-glucose, etc.
5. Nucleotides are redox cofactors:
NAD+, NADP+, FMN, FAD, Coenzyme A.

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

25. Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy
Nucleotidyl group transfer is shown here.

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

27. 10.4 What Are Nucleic Acids?
Nucleic acids are linear polymers of nucleotides linked 3' to 5' by phosphodiester bonds.
Two types:
Ribonucleic acid and
Deoxyribonucleic acid.
Know the shorthand notations.
Sequence is always read 5' to 3', left to right.
In terms of genetic information, this corresponds to "N-terminal to C-terminal“ in proteins.

28. 10.4 What Are Nucleic Acids? Figure 10.15 RNA
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.

29. 10.4 What Are Nucleic Acids? Figure 10.15 DNA:
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.

30. 10.4 What Are Nucleic Acids? Shorthand notation for DNA.
The bases are at the top.
The vertical line is the sugar numbered top to bottom.
The 5' end is to the left and the 3' end to the right.
a and b are cleavage sites for nucleases.
Linkage is a phosphodiester bond, each P has a (-) charge.

31. 10.5 What Are the Different Classes of Nucleic Acids?
DNA - one type, one purpose (three forms).
RNA - Several types, several purposes:
ribosomal RNA - the basis of structure and function of ribosomes (largest amount).
messenger RNA - carries the message for protein synthesis (fewest and unique).
transfer RNA - carries the amino acids for protein synthesis (smallest molecules).
Others:
Small nuclear RNA.
Small non-coding RNAs.
Viral

32. 10.5 What Are the Different Classes of Nucleic Acids?
Figure 10.16
The antiparallel nature of the DNA double helix.

33. The DNA Double Helix
The double helix is stabilized by hydrogen bonds and hydrophobic interactions
"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.
The hydrophobic effect from stacking of aromatic bases is also important.

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

35. The Base Pairs Postulated by Watson
Figure The Watson-Crick base pairing in A:T.
Practice drawing this structure

36. The Base Pairs Postulated by Watson
Figure The Watson-Crick base pairing in G:C.
Practice drawing this structure.

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

38. Transcription product of DNA
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
See following slides.

39. Messenger RNA Carries the Sequence Information for Synthesis of a Protein
Figure 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.

40. Messenger RNA Carries the Sequence Information for Synthesis of a Protein
In eukaryotics, mRNAs encode only one polypeptide but are more complex.

41. Eukaryotic mRNA
In eucaryotes, 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.

42. 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 the ribosomes.

43. 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.

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

45. Transfer RNAs Carry Amino Acids to Ribosomes for Use in Protein Synthesis
tRNAs are small polynucleotide chains.
73 to 94 residues each, ~10% minor bases.
Several bases are usually methylated.
Each a.a. has at least one unique tRNA which carries the a.a. to the ribosome.
The 3'-terminus carries the amino acid and the 3'-terminal sequence is always CCA-a.a.
A tRNA with an amino acid attached is called an Aminoacyl tRNA. These molecules are the substrates for protein synthesis.

46. Ribosomal RNA Provides the Structural and Functional Foundation for Ribosomes
Figure 10-23
Some unusual bases in DNA.

47. 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.

48. Why does DNA contain thymine ?
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.
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.

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

50. 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

51. 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 for mechanism.

52. 10.6 Are Nucleic Acids Susceptible to Hydrolysis?
Figure Alkaline hydrolysis of RNA. Nucleophilic attack by OH- on the P atom leads to 5'-phosphoester cleavage.

53. 10.6 Are Nucleic Acids Susceptible to Hydrolysis?
Figure Alkaline hydrolysis of RNA. Random hydrolysis of the cyclic phosphodiester intermediate gives a mixture of 2'- and 3'-nucleoside monophosphate products.

54. 10.6 Are Nucleic Acids Susceptible to Hydrolysis?
Figure Alkaline hydrolysis of RNA. The mixture of 2'- and 3'-nucleoside monophosphate products.

55. 10.6 Are Nucleic Acids Susceptible to Hydrolysis?
Figure 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.

56. 10.6 Are Nucleic Acids Susceptible to Hydrolysis?
Figure Cleavage in polynucleotide chains. Cleavage on the a-side leaves the phosphate attached to the 5'-position of the adjacent nucleotide.
Nucleic acids are cleaved by nucleases which may be “a” or “b” types. Some are exonucleases and some are endonucleases.

57. 10.6 Are Nucleic Acids Susceptible to Hydrolysis?
Figure Cleavage in polynucleotide chains.
b-side hydrolysis yields 3'-phosphate products.

58. Restriction Enzymes
Bacteria "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes". Methylation protects DNA of the bacteria.
Three known types of restriction enzymes:
Type I – has endonuclease and methylase activity and cleaves about 1000 bp from an unmethylated recognition sequence.
Type II – cleaves dsDNA only in an unmethylated recognition sequence.
Type III – has endonuclease and methylase activity and cleaves about 25 bp from an unmethylated recognition sequence.

59. Restriction Enzymes
Type II restriction enzymes cleave DNA chains at selected sites (most useful in lab).
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“.
Type II enzymes are specific and do not require ATP.
Types I and III are less specific. Type I requires ATP and Type III is ???.

60. Type II Restriction Enzymes
Recognition sites in dsDNA for Type II have a 2-fold axis of symmetry (palindromic).
Cleavage can produce staggered or "sticky" ends or "blunt” ends depending on the enzyme.
Names use 3-letter italicized code:
1st letter - genus; 2nd,3rd – species.
Following letter denotes strain.
Roman numeral = number of enzyme found.
EcoRI is the first restriction enzyme found in the R strain of E. coli.

61. Cleavage Sequences of Restriction Endonucleases

62. Cleavage Sequences of Restriction Endonucleases

63. Cleavage Sequences of Restriction Endonucleases

64. Restriction Mapping of DNA
Figure Restriction mapping analysis.

65. Restriction Mapping of DNA
Figure Restriction mapping analysis.

66. End Chapter 10 Nucleotides and Nucleic Acids