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61 genetoprotein2008

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  • To get from the chemical language of DNA to the chemical language of proteins requires 2 major stages: transcription and translation
  • eukaryotic RNA is about 10% of eukaryotic gene.
  • Beta thalassemia is an inherited blood disorder that reduces the production of hemoglobin. Symptoms of beta thalassemia occur when not enough oxygen gets to various parts of the body due to low levels of hemoglobin and a shortage of red blood cells (anemia). Signs and symptoms of thalassemia major appear in the first 2 years of life. Infants have life-threatening anemia and become pale and listless. They also have a poor appetite, grow slowly, and may develop yellowing of the skin and whites of the eyes (jaundice). The spleen, liver, and heart may be enlarged, and bones may be deformed. Adolescents with thalassemia major may experience delayed puberty. Thalassemia is a quantitative problem of too few globins synthesized, whereas sickle-cell anemia is a qualitative problem of synthesis of an incorrectly functioning globin.
  • eukaryotic RNA is about 10% of eukaryotic gene.
  • Strong evidence for a single origin in evolutionary theory.
  • The tRNA-amino acid bond is unstable. This makes it easy for the tRNA to later give up the amino acid to a growing polypeptide chain in a ribosome.
  • Walter Gilbert hypothesis: Maybe exons are functional units and introns make it easier for them to recombine, so as to produce new proteins with new properties through new combinations of domains. Introns give a large area for cutting genes and joining together the pieces without damaging the coding region of the gene…. patching genes together does not have to be so precise.

Transcript

  • 1. From Gene to Protein How Genes WorkAP Biology 2007-2008
  • 2. What do genes code for?  How does DNA code for cells & bodies?  how are cells and bodies made from the instructions in DNA DNA proteins cells bodiesAP Biology
  • 3. The “Central Dogma”  Flow of genetic information in a cell  How do we move information from DNA to proteins? n n iptio latio transcr trans DNA RNA protein trait DNA gets all the glory, replication but proteins do all the work!AP Biology
  • 4. Metabolism taught us about genes  Inheritance of metabolic diseases  suggested that genes coded for enzymes  each disease (phenotype) is caused by non-functional gene product  lack of an enzyme Am I just the sum of my proteins?  Tay sachs  PKU (phenylketonuria)  albinismmetabolic pathway disease disease disease disease A AP Biology enzyme 1 B  enzyme 2 C  enzyme 3 D  enzyme 4 E
  • 5. 1941 | 1958 Beadle & Tatum one gene : one enzyme hypothesis George Beadle Edward Tatum "for their discovery that genes act byAP Biology regulating definite chemical events"
  • 6. Beadle & Tatum X rays or ultraviolet light Wild-type Neurospora create mutations Minimal asexual spores Growth on medium complete medium positive control spores Select one of the spores Test on minimal Grow on medium to confirm complete medium presence of mutation negative control d Minimal media supplemented only with… ntifie i de n ta tioexperimentals mu Pyridoxine Choline Nucleic Arginine Riboflavin Minimalamino acid p-Amino Inositol acid Folic Niacin controlsupplements benzoic acid AP Biology acid Thiamine
  • 7. a From gene to protein a a a nucleus cytoplasm a a a a a a a transcription translation a a DNA mRNA protein a a a a a a a a a ribosome traitAP Biology
  • 8. Transcription from DNA nucleic acid language to RNA nucleic acid languageAP Biology 2007-2008
  • 9. RNA  ribose sugar  N-bases uracil instead of thymine U : A C : G  single stranded  lots of RNAs  mRNA, tRNA, rRNA, siRNA… transcriptionDNAAP Biology RNA
  • 10. Transcription  Making mRNA  transcribed DNA strand = template strand  untranscribed DNA strand = coding strand  same sequence as RNA  synthesis of complementary RNA strand  transcription bubble  enzyme coding strand  RNA polymerase G A G C A T C G T A A 3′ 5′ G A T C T C T A T A G C A T C T DNA G T A 3′ A C T G C A U C G U T G 5′ C unwinding 3′ G T A G C A rewinding mRNA 5′ RNA polymerase template strandAP Biologybuild RNA 5′→3′
  • 11. RNA polymerases  3 RNA polymerase enzymes  RNA polymerase 1  only transcribes rRNA genes  makes ribosomes  RNA polymerase 2  transcribes genes into mRNA  RNA polymerase 3  only transcribes tRNA genes  each has a specific promoter sequence it recognizesAP Biology
  • 12. Which gene is read? Promoter region  binding site before beginning of gene  TATA box binding site  binding site for RNA polymerase & transcription factors Enhancer region  binding site far upstream of gene  turns transcriptionAP Biology on HIGH
  • 13. Transcription Factors  Initiation complex  transcription factors bind to promoter region  suite of proteins which bind to DNA  hormones?  turn on or off transcription  trigger the binding of RNA polymerase to DNAAP Biology
  • 14. Matching bases of DNA & RNA A  Match RNA bases to DNA C U G bases on one of the DNA G A strands U G C U U C G A A C U A AG C U 5 RNA A 3 A C C polymerase G T G G T A C A G C T A G T C A T CG T A C CG TAP Biology
  • 15. Eukaryotic genes have junk!  Eukaryotic genes are not continuous  exons = the real gene introns  expressed / coding DNA come out!  introns = the junk  inbetween sequence intron = noncoding (inbetween) sequence eukaryotic DNA exon = coding (expressed) sequenceAP Biology
  • 16. mRNA splicing  Post-transcriptional processing  eukaryotic mRNA needs work after transcription  primary transcript = pre-mRNA  mRNA splicing  edit out introns  make mature mRNA transcript intron = noncoding (inbetween) sequence ~10,000 baseeukaryotic DNA exon = coding (expressed) sequence pre-mRNAprimary mRNA transcript ~1,000 baseAP Biology mature mRNA spliced mRNA transcript
  • 17. 1977 | 1993 Discovery of exons/introns Richard Roberts Philip Sharp adenovirus CSHL MIT common cold beta-thalassemiaAP Biology
  • 18. Splicing must be accurate  No room for mistakes!  a single base added or lost throws off the reading frame AUGCGGCTATGGGUCCGAUAAGGGCCAU AUGCGGUCCGAUAAGGGCCAU AUG|CGG|UCC|GAU|AAG|GGC|CAU Met|Arg|Ser|Asp|Lys|Gly|His AUGCGGCTATGGGUCCGAUAAGGGCCAU AUGCGGGUCCGAUAAGGGCCAU AUG|CGG|GUC|CGA|UAA|GGG|CCA|UAP Biology Met|Arg|Val|Arg|STOP|
  • 19. Whoa! I think we just broke RNA splicing enzymes a biological “rule”!  snRNPs snRNPs  small nuclear RNA snRNA  proteins exon intron exon  Spliceosome 5 3  several snRNPs  recognize splice spliceosome site sequence 5 3  cut & paste gene lariat No, not smurfs! 5 3 “snurps” exon exon mature mRNA excisedAP Biology 5 3 intron
  • 20. Alternative splicing  Alternative mRNAs produced from same gene  when is an intron not an intron…  different segments treated as exons Starting to get hard to define a gene!AP Biology
  • 21. More post-transcriptional processing  Need to protect mRNA on its trip from nucleus to cytoplasm  enzymes in cytoplasm attack mRNA  protect the ends of the molecule  add 5′ GTP cap  add poly-A tail  longer tail, mRNA lasts longer: produces more protein il ly-A ta 3 3 po A A A A A mRNA A’s 5 cap 50-250 P P 5 G PAP Biology
  • 22. a From gene to protein a a a nucleus cytoplasm a a a a a a a transcription translation a a DNA mRNA protein a a a a a a a a a ribosome traitAP Biology
  • 23. Translation from nucleic acid language to amino acid languageAP Biology 2007-2008
  • 24. How does mRNA code for proteins? DNA TACGCACATTTACGTACGCGG 4 ATCG mRNA AUGCGUGUAAAUGCAUGCGCC 4 AUCG ? protein Met Arg Val Asn Ala Cys Ala 20 How can you code for 20 amino acidsAP Biology with only 4 nucleotide bases (A,U,G,C)?
  • 25. mRNA codes for proteins in triplets DNA TACGCACATTTACGTACGCGG codon mRNA AUGCGUGUAAAUGCAUGCGCC ? proteinAP Biology Met Arg Val Asn Ala Cys Ala
  • 26. 1960 | 1968 Cracking the code Nirenberg & Khorana  Crick  determined 3-letter (triplet) codon system WHYDIDTHEREDBATEATTHEFATRAT  Nirenberg (47) & Khorana (17)  determined mRNA–amino acid match  added fabricated mRNA to test tube of ribosomes, tRNA & amino acids  created artificial UUUUU… mRNA  found that UUU coded for phenylalanineAP Biology
  • 27. Marshall Nirenberg 1960 | 1968 Har KhoranaAP Biology
  • 28. The code  Code for ALL life!  strongest support for a common origin for all life  Code is redundant  several codons for each amino acid  3rd base “wobble” Why is thewobble good?  Start codon  AUG  methionine  Stop codonsAP Biology  UGA, UAA, UAG
  • 29. How are the codons matched to amino acids? 3′ 5′ DNA TACGCACATTTACGTACGCGG 5′ 3′ mRNA AUGCGUGUAAAUGCAUGCGCC 3′ 5′ codon UAC tRNA GCA CAU anti-codon amino Met acid Arg ValAP Biology
  • 30. a From gene to protein a a a nucleus cytoplasm a a a a a a a transcription translation a a DNA mRNA protein a a a a a a a a a ribosome aa traitAP Biology
  • 31. Transfer RNA structure  “Clover leaf” structure  anticodon on “clover leaf” end  amino acid attached on 3′ endAP Biology
  • 32. Loading tRNA  Aminoacyl tRNA synthetase  enzyme which bonds amino acid to tRNA  bond requires energy  ATP → AMP  bond is unstable  so it can release amino acid at ribosome easily Trp C=O Trp C=O Trp OH H2O OH O C =O O activating enzyme tRNATrp AC C anticodon UGG mRNA tryptophan attachedAP Biology to tRNATrp tRNATrp binds to UGG condon of mRNA
  • 33. Ribosomes  Facilitate coupling of tRNA anticodon to mRNA codon  organelle or enzyme?  Structure  ribosomal RNA (rRNA) & proteins  2 subunits  large  small E P AAP Biology
  • 34. Ribosomes  A site (aminoacyl-tRNA site)  holds tRNA carrying next amino acid to be added to chain  P site (peptidyl-tRNA site)  holds tRNA carrying growing polypeptide chain Met  E site (exit site)  empty tRNA leaves ribosome 5 U AC A U G from exit site 3 E P AAP Biology
  • 35. Building a polypeptide  Initiation  brings together mRNA, ribosome subunits, initiator tRNA  Elongation  adding amino acids based on codon sequence  Termination  end codon 3 2 1 Leu Val release Ser factor Met Met Met Met Leu Leu Leu Ala Trp tRNA C A G U AC U A C G A C AA U A C GA C AA U AC G A C A A U5 CUGAA U 5 A U G CU G U 5 A U G C UG U 5 AU G C U G 3mRNA A U G 3 3 3 A CC U GG U A A E P A 3 AP Biology
  • 36. Destinations: Protein targeting secretion nucleus  Signal peptide mitochondria chloroplasts  address label cell membrane cytoplasm start of a secretory pathway etc…AP Biology
  • 37. RNA polymerase DNA Can you tell amino acids the story? exon intron tRNA pre-mRNA 5 GTP cap mature mRNA aminoacyl tRNA poly-A tail synthetase large ribosomal subunit 3 polypeptide 5 tRNA small ribosomal subunit E P AAP Biology ribosome
  • 38. The Transcriptional unit (gene?)enhancer translation translation 1000+b start exons stop 20-30b3 TAC transcriptional unit (gene) ACT 5 RNA TATA polymerase DNA DNA UTR UTR introns promoter transcription transcription start stop 5 3 pre-mRNA 5 3 AP Biology GTP mature mRNA AAAAAAAA
  • 39. Bacterial chromosome Protein Transcription Synthesis in mRNA Prokaryotes Psssst… no nucleus! Cell membrane Cell wallAP Biology 2007-2008
  • 40. Prokaryote vs. Eukaryote genes  Prokaryotes  Eukaryotes  DNA in cytoplasm  DNA in nucleus  circular  linear chromosome chromosomes  naked DNA  DNA wound on histone proteins  no introns  introns vs. exons introns come out! intron = noncoding (inbetween) sequence eukaryotic DNA exon = coding (expressed) sequenceAP Biology
  • 41. Translation in Prokaryotes Transcription & translation are simultaneous in bacteria  DNA is in cytoplasm  no mRNA editing  ribosomes read mRNA as it is being transcribedAP Biology
  • 42. Translation: prokaryotes vs. eukaryotes  Differences between prokaryotes & eukaryotes  time & physical separation between processes  takes eukaryote ~1 hour from DNA to protein  no RNA processingAP Biology
  • 43. Any Questions?? What color would a smurf turn if he held his breath?AP Biology 2007-2008
  • 44. Substitute Slides for Student Print versionAP Biology 2007-2008
  • 45. Can you tell the story?AP Biology
  • 46. The Transcriptional unitenhancer 1000+b exons 20-30b3 TAC transcriptional unit ACT 5 RNA TATA polymerase DNA introns 5 3 5 3 AP Biology