1. AP Biology 2007-2008 From Gene to Protein How Genes Work

2. AP Biology What do genes code for? proteinscellsbodies How does DNA code for cells & bodies?  how are cells and bodies made from the instructions in DNA DNA

3. AP Biology The “Central Dogma” Flow of genetic information in a cell  How do we move information from DNA to proteins? protein RNA DNAtrait DNA gets all the glory, but proteins do all the work!

4. AP Biology Inheritance of metabolic diseases  suggested that genes coded for enzymes  each disease (phenotype) is caused by non-functional gene product lack of an enzyme Tay sachs PKU (phenylketonuria) albinism Am I just the sum of my proteins? Metabolism taught us about genes ABCDE disease  enzyme 1enzyme 2enzyme 3enzyme 4 metabolic pathway

5. AP Biology Beadle & Tatum 1941 | 1958 George Beadle Edward Tatum "for their discovery that genes act by regulating definite chemical events" one gene : one enzyme hypothesis

6. AP Biology Wild-type Neurospora Minimal medium Select one of the spores Grow on complete medium Minimal control Nucleic acid Choline PyridoxineRiboflavin Arginine Minimal media supplemented only with… Thiamine Folic acid Niacin Inositol p-Amino benzoic acid Test on minimal medium to confirm presence of mutation Growth on complete medium X rays or ultraviolet light asexual spores Beadle & Tatum create mutations positive control negative control experimentals mutation identified amino acid supplements

7. AP Biology mRNA From gene to protein DNA nucleuscytoplasm a a a a a a a a a aa protein trait

8. AP Biology 2007-2008 Transcription from DNA nucleic acid language to RNA nucleic acid language

9. AP Biology RNA ribose sugar N-bases  Adenine  Guanine  Cytosine Uracil instead of Thymine lots of RNAs  mRNA, tRNA, rRNA, siRNA… RNADNA transcription

10. AP Biology Transcription Making mRNA  transcribed DNA strand = Template  untranscribed DNA strand = Coding same sequence as RNA  synthesis of complementary RNA strand transcription bubble  enzyme RNA Polymerase template strand rewinding mRNA RNA polymerase unwinding coding strand DNA C C C C C C C C CC C G G G G GG GG G G G A A A AA A A A A A A A A T T T T T T T T T T T T UU 5 3 5 3 3 5 build RNA 5  3

11. AP Biology 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 recognizes

12. AP Biology Which gene is read? Promoter  binding site before beginning of gene  TATA box  binding site for RNA polymerase & transcription factors Regulator  binding site far upstream of gene turns transcription on HIGH

13. AP Biology Transcription Factors Initiation complex  Transcription Factors suite of proteins which bind to DNA hormones? turn on or off transcription  trigger the binding of RNA polymerase to DNA

14. AP Biology Matching bases of DNA & RNA Match RNA bases to DNA bases on one of the DNA strands U AGGGGGGTTACACTTTTTCCCCAA U U U U U G G A A A CC RNA polymerase C C C C C G G G G A A A A A 5'3'

15. AP Biology Eukaryotic genes have junk! Eukaryotic genes are not continuous  Exons = the real gene expressed / coding DNA  Introns = uncoded DNA inbetween sequence eukaryotic DNA exon = coding (expressed) sequence intron = noncoding (inbetween) sequence

16. AP Biology mRNA splicing eukaryotic DNA exon = coding (expressed) sequence intron = noncoding (inbetween) sequence primary mRNA transcript mature mRNA transcript pre-mRNA spliced mRNA Post-transcriptional processing  eukaryotic mRNA needs work after transcription  5’ cap is added  Spliceosome edit out introns  Poly A Tail ~10,000 bases ~1,000 bases

17. AP Biology 1977 | 1993 Richard Roberts Philip Sharp CSHL MIT adenovirus common cold Discovery of exons/introns beta-thalassemia

18. AP Biology Splicing must be accurate No room for mistakes!  a single base added or lost throws off the Reading frame AUG|CGG|UCC|GAU|AAG|GGC|CAU AUGCGGCUAUGGGUCCGAUAAGGGCCAU AUGCGGUCCGAUAAGGGCCAU AUG|CGG|GUC|CGA|UAA|GGG|CCA|U AUGCGGCUAUGGGUCCGAUAAGGGCCAU AUGCGGGUCCGAUAAGGGCCAU Met|Arg|Ser|Asp|Lys|Gly|His Met|Arg|Val|Arg|STOP|

19. AP Biology RNA splicing enzymes snRNPs exon intron snRNA 5'3' spliceosome exon excised intron 5' 3' lariat exon mature mRNA 5' snRNP  snRNA  proteins Spliceosome  several snRNPs  recognize splice site sequence cut & paste gene

20. AP Biology Most introns start from the sequence GU and end with the sequence AG (in the 5' to 3' direction). Splice donor and Splice acceptor Another important sequence is called the branch site located 20 - 50 bases upstream of the acceptor site. The consensus sequence of the branch site is "CU(A/G)A(C/U)", where A is conserved in all genes.

21. AP Biology Example of splice sites. In over 60% of cases, the exon sequence is (A/C)AG at the donor site, and G at the acceptor site.

22. AP Biology Alternative splicing A single gene may code for more than one protein  when is an intron not an intron…  different segments treated as exons

23. AP Biology A A A A A 3' poly-A tail mRNA 5' 5' cap 3' G P P P 50-250 A’s 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 5’ cap 3’ poly A Tail

24. AP Biology mRNA From gene to protein DNA transcription nucleuscytoplasm a a a a a a a a a aa translation ribosome trait protein

25. AP Biology 2007-2008 Translation from nucleic acid language to amino acid language

26. AP Biology How does mRNA code for proteins? TACGCACATTTACGTACGCGG DNA AUGCGUGUAAAUGCAUGCGCC mRNA Met Arg Val Asn Ala Cys Ala protein ? How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)? 4 4 20 ATCG AUCG

27. AP Biology AUGCGUGUAAAUGCAUGCGCC mRNA mRNA codes for proteins in triplets TACGCACATTTACGTACGCGG DNA AUGCGUGUAAAUGCAUGCGCC mRNA Met Arg Val Asn Ala Cys Ala protein ? codon

28. AP Biology Cracking the code 1960 | 1968 Crick  determined 3-letter (triplet) codon system Nirenberg & Khorana 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 phenylalanine

29. AP Biology 1960 | 1968 Marshall Nirenberg Har Khorana

30. AP Biology 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” Start  AUG  methionine Stop  UGA, UAA, UAG

31. AP Biology How are the codons matched to amino acids? TACGCACATTTACGTACGCGG DNA AUGCGUGUAAAUGCAUGCGCC mRNA amino acid tRNA anti-codon codon 53 35 35 UAC Met GCA Arg CAU Val

32. AP Biology Inosine is a nucleoside that is formed when hypoxanthine is attached to a ribose ring via a β-N9-glycosidic bond and is a precursor to adenine.nucleosidehypoxanthineriboseglycosidic bond Inosine is commonly found in tRNAs and is essential for proper translation of the genetic code in wobble base pairs. tRNAswobble base pairs Inosine found in tRNA

33. AP Biology Degenerate code Degeneracy of the genetic code: one amino acid may be specified by more than 1 codon Example: Alanine is encoded by: GCU GCC GCA GCG 1 st position 3 rd (Wobble) position 2 nd position

34. AP Biology The Wobble Hypothesis There are four statements that form the basic Wobble hypothesis: 1. The first 2 bases of the mRNA are the major determinants and form strong base pairs with the tRNA, while the third position is wobbly. 3 2 1 Anticodon(3’) G-C-I (5’) Codon (5’) C-G-A (3’) 1 2 3 3 2 1 Anticodon(3’) G-C-I (5’) Codon (5’) C-G-U (3’) 1 2 3 3 2 1 Anticodon(3’) G-C-I (5’) Codon (5’) C-G-C (3’) 1 2 3

35. AP Biology The Wobble Hypothesis 2. The 1 st base of the anticodon pairs with the 3 rd base of the codon. a) If the 1st base of the anticodon is an A or C, then one codon is recognized. b) If the 1st base of the anticodon is a U or G, then two codons are recognized. c) If the 1st base of the anticodon is an I, then three codons are recognized (3’) X-Y-A (5’) (5’) Y-X-U (3’) (3’) X-Y-C (5’) (5’) Y-X-G (3’) (3’) X-Y-U(5’) (5’) Y-X-A (3’) (3’) X-Y-G (5’) (5’) Y-X-C (3’) (3’) X-Y-U (5’) (5’) Y-X-G (3’) (3’) X-Y-G (5’) (5’) Y-X-U(3’) (3’) X-Y-N (5’) (5’) Y-X-N (3’) anticodon codon (3’) X-Y- I (5’) (5’) Y-X-U (3’) (3’) X-Y- I (5’) (5’) Y-X-A (3’) (3’) X-Y- I (5’) (5’) Y-X-C (3’)

36. AP Biology The Wobble Hypothesis 3. If an amino acid is specified by several different codons, then the codons that differ in either of the first 2 bases require different tRNAs (3’) A-A-U (5’) (5’) U-U-A (3’) (3’) G-A-U (5’) (5’) C-U-A (3’) UUACUA Leu (3’) A-A-U (5’) (5’) A-G-U (3’) (3’) A-G-A (5’) (5’) U-C-U (3’) AGUUCU Ser

37. AP Biology The Wobble Hypothesis 4. A minimum of 32 tRNAs is required to translate all 61 codons X is either U, A, G, C Y is either U, A, G, C X-Y-G(16 combinations) X-Y-I(16 combinations) I = inosine (forms hydrogen bonds with C, U, or A)

38. AP Biology mRNA From gene to protein DNA transcription nucleuscytoplasm a a a a a a a a a aa ribosome trait protein translation

39. AP Biology Transfer RNA structure “Clover leaf” structure  anticodon on “clover leaf” end  amino acid attached on 3 end

40. AP Biology 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 Aminoacyl-tRNA synthetase anticodon tRNA Trp binds to UGG condon of mRNA Trp mRNA ACC UGG C=O OH H2OH2O O tRNA Trp tryptophan attached to tRNA Trp C=O O

41. AP Biology Ribosomes Facilitate coupling of tRNA anticodon to mRNA codon  organelle or enzyme? Structure  Large and small subunit  Made of Mainly RNA Protein EP A

42. AP Biology Ribosomes Met 5' 3' U U A C A G APE 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 E site (exit site)  empty tRNA leaves ribosome from exit site

43. AP Biology Building a polypeptide Initiation  brings together mRNA, ribosome subunits, initiator tRNA Elongation  adding amino acids based on codon sequence Termination  end codon 123 Leu tRNA Met PEA mRNA 5' 3' U U A A A A C C C AU U G G G U U A A A A C C C A U U G G G U U A A A A C C C A U U G G G U U A A A C C A U U G G G A C Val Ser Ala Trp release factor A AA CC UUGG 3'

44. AP Biology Protein targeting Specific Locations  Signal peptide and SRP  Translocation complex Destinations: secretion nucleus mitochondria chloroplasts cell membrane cytoplasm etc… start of a secretory pathway

45. AP Biology Can you tell the story?

46. AP Biology 20-30b 3' introns The Transcriptional unit transcriptional unit TACACT DNA 5' 3' 5'3' exons enhancer 1000 + b

47. AP Biology 2007-2008 Proteion Synthesis in Prokaryotes Bacterial chromosome mRNA Cell wall Cell membrane Transcription Psssst… no nucleus!

48. AP Biology Transcription & translation are simultaneous in bacteria  DNA is in cytoplasm  no mRNA editing  ribosomes read mRNA as it is being transcribed Translation in Prokaryotes

49. AP Biology Translation: prokaryotes vs. eukaryotes Differences between prokaryotes & eukaryotes  time & physical separation between processes takes eukaryote ~1 hour from DNA to protein  RNA processing

50. AP Biology Prokaryote vs. Eukaryote genes Prokaryotes  ________________ Eukaryotes  ________________ eukaryotic DNA exon = coding (expressed) sequence intron = noncoding (inbetween) sequence

51. AP Biology 2007-2008 Any Questions?? What color would a smurf turn if he held his breath?