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  • 1. Chapter 17 From Gene to Protein Rob Swatski Associate Professor of Biology HACC – York Campus
  • 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. How was the fundamental relationship between genes & proteins discovered? - examine evidence from studies of metabolic defects
  • 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. Neurospora bread mold
  • 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. EXPERIMENT Growth: No growth: Wild-type cells growing and dividing Mutant cells cannot grow and divide Minimal medium
  • 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. 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. 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. 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. 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. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide (a) Prokaryotic Cell (Bacteria)
  • 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. Nuclear envelope TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA mRNA (b) Eukaryotic cell
  • 16. Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell
  • 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. 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. 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. 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. 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. 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. 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. (a) Tobacco plant expressing a firefly gene
  • 25. (b) Pig expressing a jellyfish gene
  • 26. Synthesis of an RNA Transcript The 3 Stages of Transcription: 1. Initiation 2. Elongation 3. Termination
  • 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. Promoter: DNA sequence that RNA polymerase attaches to Transcription unit: section of DNA that is transcribed
  • 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Ribozymes Catalytic RNA molecules that act as enzymes & splice RNA - not all biological catalysts are proteins!
  • 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. 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. 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. Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide
  • 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. Amino acids Polypeptide tRNA with amino acid Ribosome attached tRNA Anticodon Codons 5 mRNA 3
  • 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. 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. 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. 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. 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. Growing polypeptide Exit tunnel tRNA molecules Large subunit E PA Small subunit 5 mRNA 3 (a) Computer model of functioning ribosome
  • 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. 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. 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. Building a Polypeptide The 3 stages of translation: 1. Initiation 2. Elongation 3. Termination All 3 stages require protein factors
  • 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. 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. 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. Amino end of polypeptide E 3 mRNA 5 P A site site GTP GDP E P A
  • 63. Amino end of polypeptide E 3 mRNA P A site site 5 GTP GDP E P A E P A
  • 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. 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. Release factor Free polypeptide 5 3 5 5 Stop codon (UAG, UAA, or UGA) 3 2 3 GTP 2 GDP
  • 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. 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. 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. 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. 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. Ribosome mRNA Signal peptide Signal peptide removed Signalrecognition particle (SRP) CYTOSOL ER LUMEN SRP receptor protein Translocation complex ER membrane Protein
  • 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. Types of Point Mutations - Base-pair substitutions - Base-pair insertions or deletions
  • 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. 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. 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. 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. 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. 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. 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. 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. 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