BIOL 102 Chp 17 PowerPoint


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BIOL 102 Chp 17 PowerPoint

  1. 1. Chapter 17 From Gene to Protein Rob Swatski Associate Professor of Biology HACC – York Campus
  2. 2. Overview: The Flow of Genetic Information DNA information = specific sequences of nucleotides DNA  protein synthesis Proteins: link genotype & phenotype Gene expression: DNA directs protein synthesis - 2 stages: transcription & translation
  3. 3. How was the fundamental relationship between genes & proteins discovered? - examine evidence from studies of metabolic defects
  4. 4. 1909: British physician Archibald Garrod 1st suggested that genes dictate phenotypes with enzymes - symptoms of inherited disease reflects inability to synthesize a certain enzyme - required understanding that cells synthesize & degrade molecules using metabolic pathways
  5. 5. Neurospora bread mold
  6. 6. Nutritional Mutants in Bread Mold George Beadle & Edward Tatum exposed Neurospora to x-rays - created mutants that could not survive on minimal medium (cannot synthesize certain molecules) Identified 3 classes of arginine-deficient mutants - each lacked a different enzyme needed to make arginine Developed the “one gene – one enzyme hypothesis” - each gene directs the synthesis of a specific enzyme
  7. 7. EXPERIMENT Growth: No growth: Wild-type cells growing and dividing Mutant cells cannot grow and divide Minimal medium
  8. 8. RESULTS Classes of Neurospora crassa Wild type Minimal medium (MM) (control) Growth Class I mutants Class II mutants Class III mutants Can grow only on citrulline or arginine Require arginine to grow No growth Condition MM  ornithine MM  citrulline MM  arginine (control) Summary of results Can grow with or without any supplements Can grow on ornithine, citrulline, or arginine
  9. 9. CONCLUSION Gene C Precursor Precursor Precursor Enzyme A Enzyme A Enzyme A Enzyme A Ornithine Ornithine Ornithine Enzyme B Enzyme B Enzyme B Enzyme B Citrulline Gene B Class II mutants (mutation in gene B) Ornithine Gene A Wild type Class I mutants (mutation in gene A) Precursor Gene (codes for enzyme) Citrulline Citrulline Citrulline Enzyme C Enzyme C Enzyme C Enzyme C Arginine Arginine Arginine Arginine Class III mutants (mutation in gene C)
  10. 10. The Products of Gene Expression: A Developing Story • Some proteins aren’t enzymes… so researchers later revised the hypothesis to “one gene – one protein” • But, many proteins consist of several polypeptides, each having its own gene Beadle & Tatum’s hypothesis is now restated as the: “one gene–one polypeptide hypothesis”
  11. 11. Basics of Transcription & Translation RNA: the bridge between genes & the proteins they code • Transcription: synthesis of RNA under the direction of DNA - produces messenger RNA (mRNA) • Translation: synthesis of a polypeptide under the direction of mRNA - ribosomes: the sites of translation
  12. 12. Eukaryotes: the nuclear envelope separates transcription from translation - eukaryotic RNA transcripts are modified via RNA processing to yield finished mRNA Prokaryotes: mRNA transcripts are immediately translated without further processing
  13. 13. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide (a) Prokaryotic Cell (Bacteria)
  14. 14. Primary transcript: initial RNA transcript from a gene before processing Central dogma: cells are governed by a cellular chain of command: DNA RNA Protein
  15. 15. Nuclear envelope TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA (b) Eukaryotic cell
  16. 16. Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell
  17. 17. The Genetic Code How are the instructions for assembling amino acids into proteins encoded into DNA? There are 20 amino acids, … but there are only 4 nucleotide bases in DNA How many bases correspond to an amino acid?
  18. 18. Codons: Triplets of Nucleotides The flow of information from gene  protein is based on a triplet code - series of non-overlapping 3-nucleotide “words” Triplet: smallest unit that can code for amino acids - “AGT” = placement of serine at its correct position in the polypeptide
  19. 19. Transcription: one of the two DNA strands (template strand) provides a pattern for ordering the nucleotide sequence in the mRNA transcript Translation: mRNA base triplets (codons) are read in the 5 to 3 direction - each codon specifies the amino acid (1 of 20) and it’s correct position in a polypeptide
  20. 20. DNA template strand 5 3 A C C A A A C C G A G T T G G T T T G G C T C A 3 5 DNA molecule Gene 1 TRANSCRIPTION Gene 2 U mRNA G G U U U G G C U C 5 A 3 Codon TRANSLATION Protein Trp Amino acid Phe Gly Ser Gene 3
  21. 21. Cracking the Code • All 64 codons were deciphered by the mid-1960s • Of the 64 triplets, 61 code for amino acids - 3 triplets are stop codons that end translation: UAA, UAG, UGA • The genetic code is redundant but not ambiguous • Codons must be read in the correct reading frame (correct groupings) in order to synthesize the specified polypeptide
  22. 22. Second mRNA base U UUU UUC UUA UCC UAC UGU UGC Cys U C UGA Stop A UCG UAG Stop UGG Trp G CUU CCU CAU CGU U CUC CCC CAC C CGC C CUA Leu Leu UCA Ser Tyr UAA Stop UUG First mRNA base (5 end of codon) Phe UAU UCU G CCA Pro CAA CUG CAG AUU A CCG ACU AAU ACC AAC AUC Ile AUA AUG ACA Met or start Thr AAA His Gln Asn Lys CGA Arg CGG AGU AGC AGA A G Ser Arg U C A AAG AGG GUU G ACG GCU GAU GGU U GUC GCC GAC GGC C GUA GUG Val GCA GCG Ala GAA GAG Asp Glu GGA GGG Gly G A G Third mRNA base (3 end of codon) U A C
  23. 23. Evolution of the Genetic Code The genetic code is shared by all living things - Genes can be transcribed & translated after being transplanted from one species to another
  24. 24. (a) Tobacco plant expressing a firefly gene
  25. 25. (b) Pig expressing a jellyfish gene
  26. 26. Synthesis of an RNA Transcript The 3 Stages of Transcription: 1. Initiation 2. Elongation 3. Termination
  27. 27. Transcription: DNA  RNA RNA synthesis is catalyzed by RNA polymerase - pries DNA strands apart - hooks RNA nucleotides together The RNA is complementary to the DNA template strand Follows same base-pairing rules as DNA - except uracil substitutes for thymine
  28. 28. Promoter: DNA sequence that RNA polymerase attaches to Transcription unit: section of DNA that is transcribed
  29. 29. Promoter Transcription unit 5 3 Start point RNA polymerase 3 5 DNA 1 Initiation 5 3 3 5 RNA transcript Unwound DNA Template strand of DNA 2 Elongation Rewound DNA 5 3 3 5 3 5 RNA transcript 3 Termination 5 3 3 5 3 5 Completed RNA transcript
  30. 30. RNA Polymerase Binding & Initiation of Transcription Promoters: signal initiation of RNA synthesis - TATA box promoter is crucial in forming the initiation complex in eukaryotes Transcription factors: needed to help bind RNA polymerase & initiate transcription Transcription initiation complex: completed assembly of transcription factors & RNA polymerase bound to a promoter
  31. 31. 1 A eukaryotic promoter Promoter Nontemplate strand DNA 5 3 3 5 T AT AAAA AT AT T T T TATA box Transcription factors Template strand Start point 2 Several transcription factors bind to DNA 5 3 3 5 3 Transcription initiation complex forms RNA polymerase II Transcription factors 5 3 5 3 RNA transcript Transcription initiation complex 3 5
  32. 32. Elongation of the RNA Strand As RNA polymerase moves along DNA, it untwists the double helix, 10-20 bases at a time - transcription rate = 40 nucleotides/sec A gene can be transcribed simultaneously by several RNA polymerases Nucleotides are added to the 3’ end of the growing RNA molecule
  33. 33. Elongation Non-template strand of DNA RNA nucleotides RNA polymerase 3 3 end 5 Direction of transcription (“downstream”) 5 Newly made RNA Template strand of DNA
  34. 34. Termination of Transcription In bacteria: RNA polymerase stops transcription at end of the terminator & the mRNA can be translated without further modification In eukaryotes: RNA polymerase continues transcription after the pre-mRNA is cleaved from the growing RNA chain - polymerase eventually falls off the DNA
  35. 35. Eukaryotic Cells Modify RNA After Transcription Enzymes in the eukaryotic nucleus modify pre-mRNA (RNA processing) before mRNA “gene” enters cytoplasm Both ends of the primary transcript are usually altered - and some interior parts of RNA are usually cut-out & other parts spliced together
  36. 36. Alteration of mRNA Ends Each end of pre-mRNA is modified in a particular way: - the 5 end gets a modified nucleotide 5 cap - the 3 end gets a poly-A tail Why Modify? - Facilitates export of mRNA - Protects mRNA from hydrolytic enzymes - Helps ribosomes attach to the 5 end
  37. 37. 5 G P P 5 Cap Protein-coding segment P Polyadenylation signal AAUAAA 5 UTR Start codon Stop codon 3 UTR 3 AAA … AAA Poly-A tail
  38. 38. Split Genes & RNA Splicing Most genes & their RNA transcripts have long noncoding regions (introns) that lie between coding regions - intron = intervening sequences (“in the way”) Exons: coding regions - expressed & translated into amino acid sequences RNA splicing: removes introns & joins exons - creates mRNA molecule with a continuous coding sequence
  39. 39. Pre-mRNA Codon numbers 5 Exon Intron Exon 5 Cap 130 31104 Intron Introns cut out and exons spliced together mRNA 5 Cap 5 UTR Poly-A tail 1146 Coding segment 3 UTR Exon 3 Poly-A tail 105 146
  40. 40. Some RNA splicing is carried out by spliceosomes - consist of a variety of proteins & small nuclear ribonucleoproteins (snRNPs = “snurps”) - snRNPs can recognize the splice sites
  41. 41. 5 RNA transcript (pre-mRNA) Intron Exon 1 Exon 2 Protein Other proteins snRNA snRNPs Spliceosome 5 Spliceosome components 5 mRNA Exon 1 Exon 2 Cut-out intron
  42. 42. Ribozymes Catalytic RNA molecules that act as enzymes & splice RNA - not all biological catalysts are proteins!
  43. 43. How can RNA function as an enzyme? - can form a 3-D structure because it can base-pair with itself - some RNA bases contain functional groups - can hydrogen-bond with other nucleic acids
  44. 44. Alternative RNA Splicing Some genes can encode more than 1 kind of polypeptide, depending on which segments are treated as exons during RNA splicing - the actual # of different proteins an organism can produce is much greater than its number of genes
  45. 45. Proteins often have a modular architecture consisting of discrete regions called domains - different exons can code for different domains in a protein Exon shuffling can result in the evolution of new proteins
  46. 46. Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide
  47. 47. Molecular Components of Translation A cell translates mRNA message into protein with the help of transfer RNA (tRNA) tRNA molecules are not identical: - each carries a specific amino acid on one end - each has an anticodon on the other end that basepairs with a complementary codon on mRNA
  48. 48. Amino acids Polypeptide tRNA with amino acid Ribosome attached tRNA Anticodon Codons 5 mRNA 3
  49. 49. The Structure & Function of tRNA 3 Amino acid attachment site 5 C Hydrogen bonds tRNA: one RNA strand, 80 nucleotides long - when flattened, it resembles a cloverleaf Anticodon
  50. 50. Can twist & fold into an “L”-shaped 3-D molecule through hydrogen-bonding Amino acid attachment site 5 3 Hydrogen bonds 5 3 Anticodon (b) 3-D structure Anticodon (c) Symbol used in this book
  51. 51. Translation requires 2 steps: “The Match Game” 1. tRNA and its amino acid are matched by the enzyme aminoacyl-tRNA synthetase - forms “charged tRNA” 2. tRNA anticodon and an mRNA codon are matched Flexible pairing at the 3rd base of a codon is called wobble - allows some tRNAs to bind to more than 1 codon
  52. 52. Aminoacyl-tRNA synthetase (enzyme) Amino acid P P P P Adenosine Adenosine P Pi ATP Pi Pi tRNA Aminoacyl-tRNA synthetase tRNA Amino acid P Adenosine AMP Computer model Aminoacyl tRNA (“charged tRNA”)
  53. 53. Ribosomes Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis - 2 ribosomal subunits (large & small) are made of proteins & ribosomal RNA (rRNA)
  54. 54. Growing polypeptide Exit tunnel tRNA molecules Large subunit E PA Small subunit 5 mRNA 3 (a) Computer model of functioning ribosome
  55. 55. P site (Peptidyl-tRNA binding site) Exit tunnel A site (AminoacyltRNA binding site) E site (Exit site) E mRNA binding site P A Large subunit Small subunit (b) Schematic model showing binding sites
  56. 56. A ribosome has 3 binding sites for tRNA: - A site: holds the tRNA carrying the next amino acid to be added to the chain - P site: holds the tRNA carrying the growing polypeptide chain - E site (Exit): where discharged tRNAs leave the ribosome
  57. 57. Growing polypeptide Amino end Next amino acid to be added to polypeptide chain tRNA 3 E mRNA 5 Codons (c) Schematic model with mRNA and tRNA
  58. 58. Building a Polypeptide The 3 stages of translation: 1. Initiation 2. Elongation 3. Termination All 3 stages require protein factors
  59. 59. Initiation of Translation Initiation stage: brings together mRNA, a tRNA with the 1st amino acid, & the 2 ribosomal subunits 1. First, the small ribosomal subunit binds with mRNA and a special initiator tRNA 2. Then the small subunit moves along mRNA until it reaches the start codon (AUG) - Initiation factors bring in the large subunit to complete the translation initiation complex
  60. 60. Large ribosomal subunit 3 U A C 5 5 A U G 3 Initiator tRNA P site GTP GDP E mRNA 5 Start codon mRNA binding site 3 Small ribosomal subunit 5 A 3 Translation initiation complex
  61. 61. Elongation of the Polypeptide Chain Elongation stage: amino acids are added one by one Each addition involves elongation factors and occurs in 3 steps: a. Codon recognition b. Peptide bond formation c. Translocation
  62. 62. Amino end of polypeptide E 3 mRNA 5 P A site site GTP GDP E P A
  63. 63. Amino end of polypeptide E 3 mRNA P A site site 5 GTP GDP E P A E P A
  64. 64. Amino end of polypeptide E 3 mRNA Ribosome ready for next aminoacyl tRNA P A site site 5 GTP GDP E E P A P A GDP GTP E P A
  65. 65. Termination of Translation Termination: - occurs when a stop codon in mRNA reaches the A site The A site accepts a release factor protein - adds a water molecule instead of an amino acid - this releases the polypeptide - the translation complex comes apart
  66. 66. Release factor Free polypeptide 5 3 5 5 Stop codon (UAG, UAA, or UGA) 3 2 3 GTP 2 GDP
  67. 67. Polyribosomes Groups of ribosomes that simultaneously translate one mRNA, forming a polyribosome (polysome) - allows a cell to quickly make many copies of a polypeptide
  68. 68. Growing polypeptides Completed polypeptide Incoming ribosomal subunits Start of mRNA (5 end) (a) End of mRNA (3 end) Ribosomes mRNA (b) 0.1 µm
  69. 69. Post-Translation A protein is usually not functional immediately after translation - requires further post-translational modification It spontaneously coils and folds into its correct 3-D shape - some activated by enzymes that cleave them - others assemble into protein subunits
  70. 70. Targeting Polypeptides to Specific Locations Two populations of ribosomes are found in cells: - Free ribosomes: synthesize proteins that function in the cytosol - Bound ribosomes: synthesize proteins on ER and those that will be secreted from the cell Ribosomes are identical and can switch from free to bound
  71. 71. Polypeptide synthesis always begins and ends in the cytosol - unless the polypeptide signals the ribosome to attach to the ER Polypeptides destined for the ER or for secretion are marked by a signal peptide - a signal-recognition particle (SRP) binds to the signal peptide - the SRP brings the signal peptide & its ribosome to the ER
  72. 72. Ribosome mRNA Signal peptide Signal peptide removed Signalrecognition particle (SRP) CYTOSOL ER LUMEN SRP receptor protein Translocation complex ER membrane Protein
  73. 73. Point Mutations - chemical changes in just 1 base pair of a gene - a change in one DNA nucleotide can lead to the production of an abnormal protein 3 5 Wild-type hemoglobin DNA C T T 5 3 G A A 3 5 mRNA 5 Mutant hemoglobin DNA C A T G T A 5 3 mRNA G A A Normal hemoglobin Glu 3 5 G U A Sickle-cell hemoglobin Val 3
  74. 74. Types of Point Mutations - Base-pair substitutions - Base-pair insertions or deletions
  75. 75. Wild type DNA template 3 strand 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end A instead of G 5 3 3 5 U instead of C 5 3 Stop Silent mutations: have no effect on the amino acid because of redundancy in the genetic code
  76. 76. Wild type DNA template 3 strand 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end T instead of C 5 3 3 5 A instead of G 3 5 Stop Missense: still codes for an amino acid, but not necessarily the right amino acid
  77. 77. Wild type DNA template 3 strand 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end A instead of T 3 5 5 3 U instead of A 5 3 Stop Nonsense: changes an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein
  78. 78. Insertions and Deletions - additions or losses of nucleotide pairs in a gene Can have a disastrous effect on the protein more often than substitutions - may produce a frameshift mutation, which alters the reading frame
  79. 79. Wild type DNA template 3 strand 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end Extra A 5 3 3 5 Extra U 5 3 Stop Frameshift Mutation causing immediate nonsense (1 base-pair insertion)
  80. 80. Wild type DNA template 3 strand 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end missing 5 3 3 5 missing 5 3 Frameshift Mutation causing extensive missense (1 base-pair deletion)
  81. 81. Wild type DNA template 3 strand 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end missing 5 3 3 5 missing 5 3 Stop No Frameshift Mutation, but one amino acid missing (3 base-pair deletion)
  82. 82. What Is a Gene? We have considered a gene as: - A discrete unit of inheritance - A region of specific nucleotide sequence in a chromosome - A DNA sequence that codes for a specific polypeptide chain In summary, a gene can be defined as: - a region of DNA that can be expressed to produce a final functional product, either a polypeptide or an RNA molecule
  83. 83. DNA TRANSCRIPTION 3 5 RNA polymerase RNA transcript RNA PROCESSING Exon RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase NUCLEUS Amino acid CYTOPLASM AMINO ACID ACTIVATION tRNA mRNA Growing polypeptide 3 A Activated amino acid P E Ribosomal subunits 5 TRANSLATION E A Codon Ribosome Anticodon