Ch3 the genetic code

2. Copyright © 2009 Pearson Education, Inc. By the end of this chapter you should be able to: • Identity the chemical components of DNA. • Assemble the double helix: The structure of DNA. • Understand the components of amino acids. • Understand the concept of genetic code. Prepared by Pratheep Sandrasaigaran

4. Copyright © 2009 Pearson Education, Inc. Nucleic Acid • Nucleic acids are produced from nucleotide polymerization. • During synthesis a series of nucleic acid condensation reactions occur between phosphate and sugar groups. Prepared by Pratheep Sandrasaigaran

5. Copyright © 2009 Pearson Education, Inc. Prepared by Pratheep Sandrasaigaran Nucleic Acid • Pentose sugar • Nitrogenous base • Phosphates • Pentose sugar • Nitrogenous base Nucleotides Nucleosides Phosphates Nucleosides+ Nucleotides=

10. Copyright © 2009 Pearson Education, Inc. DNA vs RNA Bases • The nucleotide bases in nucleic acids contain nitrogen derived from either purines or pyrimidines. • Purines (Double ring) • Adenine • Guanine • Pyrimidines (Single ring) • Cytosine • Thymine* • Uracil* Prepared by Pratheep Sandrasaigaran

14. Copyright © 2009 Pearson Education, Inc. DNA is double helix • Discovered by Watson and Crick in 1953 • Base composition analysis of hydrolyzed samples of DNA • X-ray diffraction studies of DNA. • Adenine and thymine pair via two hydrogen bonds between opposing strands. • Guanine and cytosine pair via three hydrogen bonds. • Base pairing results in two complementary polynucleotides, which run antiparallel to each other. Prepared by Pratheep Sandrasaigaran

16. Copyright © 2009 Pearson Education, Inc. RNA is Single strand- mRNA • Messenger RNA • Carries genetic information from the nucleus into the cytoplasm. • In eukaryotes, it is derived by splicing the initial RNA transcript (heteronuclear RNA that holds introns) Prepared by Pratheep Sandrasaigaran

18. Copyright © 2009 Pearson Education, Inc. RNA is Single strand- tRNA • Transfer RNA • Linear molecule with an average of 76 nucleotides • Exhibits extensive intramolecular base pairing, giving it a ‘clover-leaf’-shaped secondary structure • Carries specific amino acids to the site of protein synthesis. Prepared by Pratheep Sandrasaigaran

19. Copyright © 2009 Pearson Education, Inc. RNA is Single strand- tRNA Prepared by Pratheep Sandrasaigaran Active sites on the acceptor arm • Terminal CCA group can accept a specific amino acid. • anticodon arm, recognizes the corresponding mRNA codon. • Specific base pairing within the five arms helps to maintain the secondary structure. Active sites on the anticodon arm Diagram adopted from Crash course: Cell Biology and Genetics, 4th ED

20. Copyright © 2009 Pearson Education, Inc. RNA is Single strand- rRNA • Ribosomal RNA (ribosomal component) • In a eukaryotic cell each ribosome consists of two unequal subunits, made up of proteins and RNA, called the S (small) and L (large) subunits held together by magnesium ions. • The RNA molecules undergo extensive intramolecular base pairing, which determines the ribosomal structure. Prepared by Pratheep Sandrasaigaran

22. Copyright © 2009 Pearson Education, Inc. 1. What are nucleic acids? • DNA and RNA, the nucleic acids, are the molecules responsible for the hereditary information that commands the protein synthesis in living beings. Prepared by Pratheep Sandrasaigaran 2. What are the units nucleic acids constituted? What are the chemical entities that compose that unit? • Nucleic acids are formed by sequences of nucleotides • Nucleotides are constituted by one molecule of sugar (deoxyribose in DNA and ribose in RNA) bound to one molecule of phosphate and to one nitrogen-containing base 3. Which two groups can the nitrogen-containing bases that form DNA and RNA be classified? • The nitrogen-containing bases that form DNA and RNA are classified as pyrimidine and purine bases

23. Copyright © 2009 Pearson Education, Inc. 4. What is the criterion used in purine-pyrimidine classification? • Cytosine, thymine and uracil, have only one nitrogenized carbon ring. The others, adenine and guanine, have two nitrogenized associated carbon rings Prepared by Pratheep Sandrasaigaran 5. Which type of chemical bond maintains the pairing of each chain in the DNA molecule? • To form the DNA molecule, purine bases bind to pyrimidine bases by intermolecular bonds called hydrogen bonds. Hydrogen bonds occur when there is hydrogen near one of these electronegative elements: fluorine, oxygen or nitrogen

24. Copyright © 2009 Pearson Education, Inc. 6. What is the completing sequence of nitrogen- containing bases for a AGCCGTTAAC fragment of a DNA chain? Prepared by Pratheep Sandrasaigaran 7. What are the three main types of RNA? • Messenger RNA, or mRNA, transfer RNA, or tRNA, and ribosomal RNA, or rRNA, are the three main types of RNA. • TCGGCAATTG 8. What is meant by heterogeneous RNA? • The newly formed RNA molecule, a precursor of mRNA, is called heterogeneous RNA (hnRNA). The heterogenous RNA bears portions called introns and portions called exons. The hnRNA is processed in many chemical steps, introns are removed and mRNA is created formed only of exons, the biologically active nucleotide sequences

25. Copyright © 2009 Pearson Education, Inc. 9. Concerning their biological function what is the difference between DNA and RNA? Prepared by Pratheep Sandrasaigaran 10. Is there any situation in which DNA is made based on a RNA template? • The process in which DNA is synthesized having as template a RNA chain is called reverse transcription. In cells infected by retroviruses (RNA viruses, like the AIDS or SARS viruses) reverse transcription occurs and DNA is made from information contained in the viral RNA. Viral RNA within the host cell produces DNA with the help of an enzyme called reverse transcriptase. • DNA is the source of information for RNA production (transcription) and thus for protein synthesis. DNA is still the basis of heredity due to its replication capability

27. Copyright © 2009 Pearson Education, Inc. Evidence 1: Base-Composition Studies • Between 1949 and 1953, Erwin Chargaff and his colleagues used chromatographic methods to separate the four bases in DNA samples from various organisms. Prepared by Pratheep Sandrasaigaran Diagram adopted from William S and Klug. Concept of Genetics, 10th ED

28. Copyright © 2009 Pearson Education, Inc. What can be derived from Erwin Chargaff? • The amount of adenine residues is proportional to the amount of thymine residues in DNA. • The amount of guanine residues is proportional to the amount of cytosine residues. • The sum of the purines (A + G) equals the sum of the pyrimidines (C + T). • The percentage of (G + C) does not necessarily equal the percentage of (A + T). Prepared by Pratheep Sandrasaigaran

29. Copyright © 2009 Pearson Education, Inc. Evidence 2: X-Ray Diffraction Analysis • When fibers of a DNA molecule are subjected to X-ray bombardment, the X rays scatter (diffract) in a pattern that depends on the molecule’s atomic • William Astbury (1938) detected a periodicity 3.4 angstroms (3.4-Å) • Bases were stacked like coins on top of one another. Prepared by Pratheep Sandrasaigaran

30. Copyright © 2009 Pearson Education, Inc. Evidence 2: X-Ray Diffraction Analysis • Between 1950 and 1953, Rosalind Franklin suggested that the structure of DNA was some sort of helix. Prepared by Pratheep Sandrasaigaran • X-ray diffraction photograph by Rosalind Franklin using the B form of purified DNA fibers. Diagram adopted from William S and Klug. Concept of Genetics, 10th ED

31. Copyright © 2009 Pearson Education, Inc. The Watson–Crick Model • Two long polynucleotide chains are coiled around a central axis, forming a right- handed double helix. • The two chains are antiparallel; that is, their C-5’-to-C-3’orientations run in opposite directions. • The bases of both chains are flat structures lying perpendicular to the axis; they are “stacked” on one another, 3.4 Å (0.34 nm) apart, on the inside of the double helix. Prepared by Pratheep Sandrasaigaran Diagram adopted from Crash course: Cell Biology and Genetics, 4th ED

32. Copyright © 2009 Pearson Education, Inc. The Watson–Crick Model • The nitrogenous bases of opposite chains are paired as the result of the formation of hydrogen bonds • In DNA, only A -T and G-C pairs occur. • Each complete turn of the helix is 34 Å (3.4 nm) long; thus, each turn of the helix is the length of a series of 10 base pairs. Prepared by Pratheep Sandrasaigaran Diagram adopted from Crash course: Cell Biology and Genetics, 4th ED

33. Copyright © 2009 Pearson Education, Inc. The Watson–Crick Model • A larger major groove alternating with a smaller minor groove winds along the length of the molecule. • The double helix has a diameter of 20 Å (2.0 nm). Prepared by Pratheep Sandrasaigaran Diagram adopted from Crash course: Cell Biology and Genetics, 4th ED

34. Copyright © 2009 Pearson Education, Inc. DNA and Inheritance • The structure of DNA is a linear sequence of deoxyribonucleotides. • There are regions within the DNA that contain protein-coding genes. • How is the information within DNA decoded for translation of proteins? • Central dogma – flow of information from Prepared by Pratheep Sandrasaigaran Diagram adopted from The Facts On File Illustrated Guide to the Human Body: Cells and Genetics

35. Copyright © 2009 Pearson Education, Inc. Transcription & translation • Information present on one of the two strands of DNA is transferred into an RNA complement in a process called transcription. • Messenger RNA (mRNA) serves as a messenger molecule transporting coded information out of the nucleus. • mRNAs associate with ribosomes, where translation occurs. Prepared by Pratheep Sandrasaigaran

38. Copyright © 2009 Pearson Education, Inc. 1. Who were James Watson, Francis Crick and Maurice Wilkins • Watson (North American), Crick (British) and Wilkins (New Zealander) were the discoverers of the molecular structure of DNA, the double helix made of two polynucleotide chains paired by their nitrogen-containing bases. They won the Nobel prize in Medicine in 1962 for the discovery. Prepared by Pratheep Sandrasaigaran 2. What can you conclude from the table below? Diagram adopted from William S and Klug. Concept of Genetics, 10th ED

40. Copyright © 2009 Pearson Education, Inc. AMINO ACIDS • Amino acids are the subunits of proteins, and they all have the same basic structure. • A central carbon atom (the a carbon) • An amino (NH2) group at the a carbon • A carboxyl group (COOH) • A side group (R). Prepared by Pratheep Sandrasaigaran Diagram adopted from Crash course: Cell Biology and Genetics, 4th ED

41. Copyright © 2009 Pearson Education, Inc. AMINO ACIDS • There are 20 naturally occurring amino acids, which differ in their side group. • All amino acids, except glycine, have an asymmetrical α-carbon atom, giving rise to D or L stereoisomer forms; however, only the L form is found in humans. • Amino acids form proteins by joining together through peptide bonds. Prepared by Pratheep Sandrasaigaran Diagram adopted from Crash course: Cell Biology and Genetics, 4th ED

42. Copyright © 2009 Pearson Education, Inc. Amino acids code Nonpolar and uncharged A Ala Alanine F Phe Phenylalanine G Gly Glycine I Ile Isoleucine L Leu Leucine M Met Methionine P Pro Proline V Val Valine W Trp Tryptophan Polar and uncharged C Cys Cysteine N Asn Asparagine Q Gln Glutamine S Ser Serine T Thr Threonine Y Tyr Tyrosine Positively charged (basic) H His Histidine K Lys Lysine R Arg Arginine Negatively charged (acidic) D Asp Aspartic acid E Glu Glutamic acid Ambiguous codes B Asx Asparagine or aspartic acid Z Glx Glutamine or glutamic acid Amino acids code

43. Copyright © 2009 Pearson Education, Inc. Prepared by Pratheep Sandrasaigaran Amino acids code • The three-dimensional structure of a protein and, therefore, its behavior, is determined by the characteristics and interactions of the side chains in its amino- acid sequence • Links between amino-acid residues occur through hydrogen bonds, disulphide bridges, hydrophobic bonds and ionic bonds; to stabilize the protein in its characteristic conformation.

44. Copyright © 2009 Pearson Education, Inc. Links between amino-acid residues • Hydrogen bonds occur between carbonyl (C-O) and amino (N–H) groups. • Disulphide bridges are covalent bonds between -SH groups of cysteine residues. • Non-covalent hydrophobic bonds form between two hydrophobic residues. • Electrovalent (ionic) bonds occur between a negative group of one amino-acid residue and a positive group of another amino-acid residue. Prepared by Pratheep Sandrasaigaran

46. Copyright © 2009 Pearson Education, Inc. No 1 L 3 L Amino acid 1 A Ala Alanine 2 F Phe Phenylalanine 3 G Gly Glycine 4 I Ile Isoleucine 5 L Leu Leucine 6 M Met Methionine 7 P Pro Proline 8 V Val Valine 9 W Trp Tryptophan 10 C Cys Cysteine 11 N Asn Asparagine 12 Q Gln Glutamine 13 S Ser Serine 14 T Thr Threonine 15 Y Tyr Tyrosine 16 H His Histidine 17 K Lys Lysine 18 R Arg Arginine 19 D Asp Aspartic acid 20 E Glu Glutamic acid Prepared by Pratheep Sandrasaigaran

48. Copyright © 2009 Pearson Education, Inc. Figure 14.3 Deciphering the Code • Marshall Nirenberg and Heinrich Matthaei at the National Institutes of Health used a precise and logical series of experiments to “crack the code. • They were among the first to characterize specific coding sequences. • Made possible by advancements that: • Allowed protein synthesis in vitro • Synthesizing RNA strands in vitro Prepared by Pratheep Sandrasaigaran Diagram adopted from Human Genetics concepts and Application 9th ed

49. Copyright © 2009 Pearson Education, Inc. 8 Characteristics of the Genetic Code 1. Written in linear form of ribonucleotide bases (mRNA). 2. Each word consists of 3 ribonucleotide letters which (triplet code- codon) specifies one amino acid. 3. The code is unambiguous- each triplet specifies only a single amino acid. 4. The code is degenerate, given amino acid can be specified by more than one triplet codon. Prepared by Pratheep Sandrasaigaran

50. Copyright © 2009 Pearson Education, Inc. 5. The code is commaless; Once translation of mRNA begins, the codons are read one after the other with no breaks between them (until a stop signal is reached). 6. The code contains 1 start and 3 stop codons 7. The code is non-overlapping 8. The code is (nearly) universal. With only minor exceptions, a single coding dictionary is used by almost all viruses, prokaryotes, archaea, and eukaryotes. Prepared by Pratheep Sandrasaigaran 8 Characteristics of the Genetic Code

51. Copyright © 2009 Pearson Education, Inc. The Genetic Code Uses Ribonucleotide Bases as “Letters” Prepared by Pratheep Sandrasaigaran • Even though genetic information is stored in DNA, the code that is translated into proteins resides in RNA. • How only four nucleotides could specify 20 the amino acids? Diagram adopted from William S and Klug. Concept of Genetics, 10th ED

52. Copyright © 2009 Pearson Education, Inc. Evidence for the Triplet Code • How Many RNA Bases Specify One amino acid code; 20 amino acids code? • If a codon consisted of only one mRNA base? • Two base, for example, provides only 16 unique code words (42 ). Not enough..! • A triplet code yields 64 words (43 ) and therefore is sufficient for the 20 amino acids. • A four-letter code (44 ), which would specify 256 words. Prepared by Pratheep Sandrasaigaran Theory

53. Copyright © 2009 Pearson Education, Inc. Evidence for the Triplet Code • Experimental work of Francis Crick, Leslie Barnett, Brenner and R. J. Watts-Tobin provided the solid evidence for a triplet code. • The insertion of a single nucleotide shifts all subsequent triplets out of the reading frame (frameshift mutations). • They exposed the chemicals that add or remove one, two, or three bases at the rII locus of bacteriophage T4 and examine their reproduction on E. coli K12 (viral gene is well known). Prepared by Pratheep Sandrasaigaran

54. Copyright © 2009 Pearson Education, Inc. Evidence for the Triplet Code • Alteration in DNA sequence happen due to disruption of the reading frame. • However, continues experiment shows no huge variation in the protein production and the reproduction ability of T4 phage… WHY? • The result was a protein with a stretch of the wrong amino acids, like a sentence with a few words in the middle that are misspelled Prepared by Pratheep Sandrasaigaran Diagram adopted from Human Genetics concepts and Application 9th ed

55. Copyright © 2009 Pearson Education, Inc. Evidence for a Non-Overlapping Code • If code was overlapping: • Amino acid sequences would be restricted- Sydney Brenner • Base substitutions would affect two adjacent amino acids- tobacco mosaic virus (TMV) and human hemoglobin • Translation would be too complex to be efficient- Francis Crick Prepared by Pratheep Sandrasaigaran

56. Copyright © 2009 Pearson Education, Inc. Evidence for a Non-Overlapping Code • Consider a hypothetical mRNA sequence: AUGCCCAAG • If the genetic code is triplet and a DNA sequence is “read” in a nonoverlapping manner, then this sequence has only three codons and specifies three amino acids. • Do you agree? • However, if the DNA sequence is overlapping, the sequence specifies seven codons. Prepared by Pratheep Sandrasaigaran

57. Copyright © 2009 Pearson Education, Inc. Evidence for a Non-Overlapping Code • Even though the genetic code is non-overlapping, it is possible to read any DNA or RNA sequence in three different reading frames, depending upon the “start” base. Prepared by Pratheep Sandrasaigaran Diagram adopted from Human Genetics concepts and Application 9th ed

58. Copyright © 2009 Pearson Education, Inc. Evidence for a Commaless and Degnerate Code • Crick hypothesized, on the basis of genetic evidence, that the code would be commaless. • Only 20 of the 64 possible codons would specify an amino acid and that the remaining 44 would carry no coding assignment. What do you think? • More than one codon specifies the same amino acid. • Crick’s frameshift studies suggest that wild-type function is restored when there is, (+) with (-); (++) with (- -); and (+++) with (- - -); the original frame of reading is restored. Prepared by Pratheep Sandrasaigaran

59. Copyright © 2009 Pearson Education, Inc. The code contains 1 start and 3 stop codons • Chemical analysis eventually showed that the genetic code includes directions for starting and stopping translation. • The codon AUG signals “start,” and the codons UGA, UAA, and UAG signify “stop.” Prepared by Pratheep Sandrasaigaran AUG UGA UAA UAG

60. Copyright © 2009 Pearson Education, Inc. The code is (nearly) universal • All life evolved from a common ancestor, hence all species use the same mRNA codons to specify the same amino acids. • Do you think there is exceptions to the universality of the genetic code? • Mitochondria • Certain single-celled eukaryotes (ciliated protozoa) • These deviations may be tolerated because they do not affect the major repositories of DNA. Prepared by Pratheep Sandrasaigaran

62. Copyright © 2009 Pearson Education, Inc. The code is unambiguous • Which codons specify which amino acids? • Marshall Nirenberg and Heinrich Matthaei experiments. Prepared by Pratheep Sandrasaigaran Diagram adopted from Human Genetics concepts and Application 9th ed

63. Copyright © 2009 Pearson Education, Inc. Marshall Nirenberg and Heinrich Matthaei experiments • Synthesized simple mRNA molecules were added to test tubes that contained all the chemicals and structures needed for translation, extracted from E. coli. • The 1st synthetic mRNA they made had the sequence UUUUUU…. Result: Peptides of phenylalanine. • Conclusion: The codon UUU specifies the amino acid phenylalanine Prepared by Pratheep Sandrasaigaran

64. Copyright © 2009 Pearson Education, Inc. Marshall Nirenberg and Heinrich Matthaei experiments • The 2nd experiments AAAAAA….. Result: Peptide of lysine • The 3rd experiments CCCCCC…. Result: Peptide for Proline. • GGGGGG…. was unstable, so this part of the experiment could not be done. • Next to prove other codon-amino acid pairs hence researchers synthesized chains of alternating bases Prepared by Pratheep Sandrasaigaran

65. Copyright © 2009 Pearson Education, Inc. Marshall Nirenberg and Heinrich Matthaei experiments • Synthetic mRNA of sequence AUAUAU . . . introduced codons AUA and UAU alternating Isoleucines and Tyrosines, but which one is which? • Another experiment with a more complex sequence answered the question. • The mRNA UUUAUAUUUAUA, UUU codes for phenylalanine, AUA code for isoleucine. • Hence if AUA codes for isoleucine, then UAU must code for tyrosine… right? Prepared by Pratheep Sandrasaigaran

66. Copyright © 2009 Pearson Education, Inc. The code is unambiguous • Sixty of the possible 64 codons specify particular amino acids • Three indicate “stop,” and one encodes both the amino acid methionine and “start.” • This means that some amino acids are specified by more than one codon. • For example, both UUU and UUC encode phenylalanine. Prepared by Pratheep Sandrasaigaran

68. Copyright © 2009 Pearson Education, Inc. The Genetic code • Almost all amino acids are specified by two, three, or four different codons. • Three amino acids (arginine, serine, and leucine) are specified by six codons. • Methionine and tryptophan are encoded by single codons. • In many codons specifying the same amino acid (synonymous codons), the first two positions are the same but the third position differs (Proline); degenerate Prepared by Pratheep Sandrasaigaran

69. Copyright © 2009 Pearson Education, Inc. Wobble hypothesis • Crick postulated the wobble hypothesis. • He predicted that the initial two ribonucleotides of triplet codes are often more critical than the third member in attracting the correct tRNA. • He hypothesized that hydrogen bonding at the third position of the codon-anticodon interaction would be less spatially constrained. • In addition, it may also need not to adhere strictly to the base-pairing rules. Prepared by Pratheep Sandrasaigaran

70. Copyright © 2009 Pearson Education, Inc. Wobble hypothesis • 61 different tRNAs could theoretically exist, one for each codon that specifies an amino acid, however only 49 different genes encode tRNAs. • This is because the same type of tRNA can detect synonymous codons that differ only in whether the wobble (third) position is U or C. • The same type of tRNA, for example, binds to both UUU and UUC codons, which specify the amino acid phenylalanine. • Synonymous codons ending in A or G use different tRNAs Prepared by Pratheep Sandrasaigaran

71. Copyright © 2009 Pearson Education, Inc. • Written in linear form • Each word consists of 3 ribonucleotide letters • The code is unambiguous • The code is degenerate • The code contains 1 start and 3 stop codons • The code is commaless • The code is non-overlapping • The code is (nearly) universal Summary- The Genetic Code Prepared by Pratheep Sandrasaigaran

73. Copyright © 2009 Pearson Education, Inc. • Written in linear form • Each word consists of 3 ribonucleotide letters • The code is unambiguous • The code is degenerate • The code contains 1 start and 3 stop codons • The code is commaless • The code is non-overlapping • The code is (nearly) universal Describe the characteristics of Genetic code Prepared by Pratheep Sandrasaigaran

74. Copyright © 2009 Pearson Education, Inc. 1. In the film Jurassic Park, which is about cloned dinosaurs, a cartoon character named Mr. DNA talks about the billions of genetic codes in DNA. Why is this statement incorrect? Prepared by Pratheep Sandrasaigaran 2. Titin is a muscle protein named for its size. its gene has the largest known coding sequence of 80,781 DNA bases. How many amino acids long is it? 3. An extraterrestrial life form has a triplet genetic code with five different bases. How many different amino acids can this code specify, assuming no degeneracy?

75. Copyright © 2009 Pearson Education, Inc. 4. Explain the work of Francis Crick at al. on how did they prove a codon is triplet. Prepared by Pratheep Sandrasaigaran 5. What is Marshall Nirenberg and Heinrich Matthaei experiments is about? Explain in details. 6. What is a Wobble hypothesis?