Frederick Griffith (1928)

1. Frederick Griffith (1928) Conclusion: living R bacteria transformed into deadly S bacteria by unknown, heritable substance Oswald Avery, et al. (1944) • Discovered that the transforming agent was DNA

2. Hershey and Chase (1952) • Bacteriophages: virus that infects bacteria; composed of DNA and protein Protein = radiolabel S DNA = radiolabel P Conclusion: DNA entered infected bacteria  DNA must be the genetic material!

3. Edwin Chargaff (1947) Chargaff’s Rules: • DNA composition varies between species • Ratios: • %A = %T and %G = %C

4. Structure of DNA Scientists: • Watson & Crick • Rosalind Franklin DNA = double helix • “Backbone” = sugar + phosphate • “Rungs” = nitrogenous bases

5. Structure of DNA Nitrogenous Bases • Adenine (A) • Guanine (G) • Thymine (T) • Cytosine (C) • Pairing: • purine + pyrimidine • A = T • G Ξ C purine pyrimidine

6. Structure of DNA Hydrogen bondsbetween base pairs of the two strands hold the molecule together like a zipper.

7. Structure of DNA Antiparallel: one strand (5’ 3’), other strand runs in opposite, upside-down direction (3’  5’)

10. DNA Comparison Prokaryotic DNA Eukaryotic DNA Double-stranded Linear Usually 1+ chromosomes In nucleus DNA wrapped around histones (proteins) Forms chromatin • Double-stranded • Circular • One chromosome • In cytoplasm • No histones • Supercoiled DNA

11. Replication is semiconservative

13. Major Steps of Replication: • Helicase:unwinds DNA at origins of replication • Initiation proteins separate 2 strands  forms replication bubble • Primase: puts down RNA primer to start replication • DNA polymerase III: adds complimentary bases to leading strand (new DNA is made 5’ 3’) • Lagging strand grows in 3’5’ direction by the addition of Okazaki fragments • DNA polymerase I: replaces RNA primers with DNA • DNA ligase: seals fragments together

14. 1. Helicase unwinds DNA at origins of replication and creates replication forks

15. 3. Primase adds RNA primer

16. 4. DNA polymerase III adds nucleotides in 5’3’ direction on leading strand

17. Replication on leading strand

18. Leading strand vs. Lagging strand

19. Okazaki Fragments: Short segments of DNA that grow 5’3’ that are added onto the Lagging Strand DNA Ligase: seals together fragments

22. Proofreading and Repair • DNA polymerases proofread as bases added • Mismatch repair: special enzymes fix incorrect pairings • Nucleotide excision repair: • Nucleases cut damaged DNA • DNA poly and ligase fill in gaps

23. Nucleotide Excision Repair Errors: • Pairing errors: 1 in 100,000 nucleotides • Complete DNA: 1 in 10 billion nucleotides

24. Problem at the 5’ End • DNA poly only adds nucleotides to 3’ end • No way to complete 5’ ends of daughter strands • Over many replications, DNA strands will grow shorter and shorter

25. Telomeres: repeated units of short nucleotide sequences (TTAGGG) at ends of DNA • Telomeres “cap” ends of DNA to postpone erosion of genes at ends (TTAGGG) • Telomerase: enzyme that adds to telomeres • Eukaryotic germ cells, cancer cells Telomeres stained orange at the ends of mouse chromosomes

26. Flow of genetic information • Central Dogma: DNA  RNA  protein • Transcription: DNA  RNA • Translation: RNA  protein • Ribosome = site of translation • Gene Expression: process by which DNA directs the synthesis of proteins (or RNAs)

27. Flow of Genetic Information in Prokaryotes vs. Eukaryotes

28. one gene = one polypeptide DNA RNA Nucleic acid composed of nucleotides Single-stranded Ribose=sugar Uracil Helper in steps from DNA to protein • Nucleic acid composed of nucleotides • Double-stranded • Deoxyribose=sugar • Thymine • Template for individual

29. RNA plays many roles in the cell • pre-mRNA=precursor to mRNA, newly transcribed and not edited • mRNA= the edited version; carries the code from DNA that specifies amino acids • tRNA= carries a specific amino acid to ribosome based on its anticodon to mRNA codon • rRNA= makes up 60% of the ribosome; site of protein synthesis • snRNA=small nuclear RNA; part of a spliceosome. Has structural and catalytic roles • srpRNA=a signal recognition particle that binds to signal peptides • RNAi= interference RNA; a regulatory molecule

30. The Genetic Code For each gene, one DNA strand is the template strand mRNA (5’ 3’) complementary to template mRNA triplets (codons) code for amino acids in polypeptide chain

31. The Genetic Code 64 different codon combinations Redundancy: 1+ codons code for each of 20 AAs Reading frame: groups of 3 must be read in correct groupings This code is universal: all life forms use the same code.

32. Transcription Transcription unit: stretch of DNA that codes for a polypeptide or RNA (eg. tRNA, rRNA) RNA polymerase: • Separates DNA strands and transcribes mRNA • mRNA elongates in 5’ 3’ direction • Uracil (U) replaces thymine (T) when pairing to adenine (A) • Attaches to promoter (start of gene) and stops at terminator (end of gene)

33. 1. Initiation Bacteria: RNA polymerase binds directly to promoter in DNA

34. 1. Initiation Eukaryotes: TATA box = DNA sequence (TATAAAA) upstream from promoter Transcription factors mustrecognize TATA box before RNA polymerase can bind to DNA promoter

35. 2. Elongation • RNA polymerase adds RNA nucleotides to the 3’ end of the growing chain (A-U, G-C)

36. 2. Elongation As RNA polymerase moves, it untwists DNA, then rewinds it after mRNA is made

37. 3. Termination RNA polymerase transcribes a terminatorsequence in DNA, then mRNA and polymerase detach. It is now called pre-mRNA for eukaryotes. Prokaryotes = mRNA ready for use

38. Additions to pre-mRNA: • 5’ cap(modified guanine) and 3’poly-A tail(50-520 A’s)are added • Help export from nucleus, protect from enzyme degradation, attach to ribosomes

39. RNA Splicing • Pre-mRNA has introns (noncoding sequences) and exons (codes for amino acids) • Splicing = introns cut out, exons joined together

40. RNA Splicing • small nuclear ribonucleoproteins = snRNPs • snRNP = snRNA + protein • Pronounced “snurps” • Recognize splice sites • snRNPs join with other proteins to form a spliceosome Spliceosomescatalyze the process of removing introns and joining exons Ribozyme = RNA acts as enzyme

41. Why have introns? • Some regulate gene activity • Alternative RNA Splicing: produce different combinations of exons • One gene can make more than one polypeptide! • 20,000 genes  100,000 polypeptides

42. Components of Translation • mRNA = message • tRNA= interpreter • Ribosome = site of translation

43. tRNA • Transcribed in nucleus • Specific to each amino acid • Transfer AA to ribosomes • Anticodon: pairs with complementary mRNA codon • Base-pairing rules between 3rd base of codon & anticodon are not as strict. This is called wobble.

44. tRNA • Aminoacyl-tRNA-synthetase: enzyme that binds tRNA to specific amino acid

45. Ribosomes • Ribosome = rRNA + proteins • made in nucleolus • 2 subunits

46. Ribosomes Active sites: • A site: holds AA to be added • P site: holds growing polypeptide chain • E site: exit site for tRNA

47. Translation:1. Initiation • Small subunit binds to start codon (AUG) on mRNA • tRNA carrying Met attaches to P site • Large subunit attaches

48. 2. Elongation

49. 3.Termination • Stop codon reached and translation stops • Release factor binds to stop codon; polypeptide is released • Ribosomal subunits dissociate

50. Polyribosomes • A single mRNA can be translated by several ribosomes at the same time