Frederick Griffith: Transformation Conclusion: bacteria could give other bacteria heritable traits, even after they were dead.

Transcription

1 Frederick Griffith: Transformation 1928 Conclusion: bacteria could give other bacteria heritable traits, even after they were dead. 1

2 Avery, McCarty & MacLeod: Griffiths Refined (1944) Refined Griffith's Experiment Exposed R-strain Streptococcus to purified S-strain protein, and purified S-strain DNA DNA was transforming agent 2

3 Hershey & Chase: The "Blender" Experiment (1952) Worked with bacteriophages virus that infects bacteria Protein = radiolabel S DNA = radiolabel P 3

4 Conclusion: DNA entered infected bacteria DNA must be the genetic material! 4

5 DNA - Deoxyribose nucleic acid Composed of nucleotides 2 primary functions 1. Control protein (enzyme) production 2. Duplicate itself for new cells that are created undergo changes (mutate) 5

6 Nucleotide Deoxyribose sugar Phosphate group Nitrogenous base A, T, C, G 6

7 4 Bases Nitrogenous Bases Adenine (A), Thymine (T), Cytosine (C), Guanine (G) Purine Double ring A and G Pyrimidine single ring T and C 7

8 Chargaff s Rule DNA composition varies between species Chargaff s rule: C=G A=T 8

9 Rosalind Franklin (1950 s) Worked with Maurice Wilkins X-ray crystallography = images of DNA Provided measurements on chemistry of DNA

10 James Watson & Francis Crick (1953) Discovered the double helix by building models to conform to Franklin s X-ray data and Chargaff s Rules.

11 DNA is a Double Helix Sugar and phosphate form the backbone covalently bonded to each other ("phosphodiester bonds") Bases lie between the backbone Nucleotides Held together by H-bonds between the bases A-T 2 H bonds G-C 3 H bonds 11

12 Orientation of DNA Phosphate end is the 5' end The opposite end (sugar) is the 3' end Q: Since DNA is complementary, what end matches with the 5 end? 12

13 DNA Comparison Prokaryotic DNA Double-stranded Circular One chromosome In cytoplasm Supercoiled DNA (nucleoid) No histones Eukaryotic DNA Double-stranded Linear Usually 1+ chromosomes In nucleus Chromatin = DNA wrapped around histones (proteins)

14 Plasmids small, circular, double-stranded DNA molecule Different from chromosomal DNA naturally exist in bacterial cells Often provide bacteria with genetic advantages antibiotic resistance Hundreds to thousands of base pairs each daughter cell receives a copy of plasmid Bacteria can also transfer plasmids to one another through conjugation 14

15 Central Dogma "Central Dogma": Term coined by Francis Crick to explain how information flows in cells.

16 Semiconservative Replication = each strand has 1 parent and 1 new strand

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18 1. DNA Replication 1. DNA helicase attaches to DNA molecule and unzips DNA into two strands 1. Single strand binding proteins keep it open 2. Topoisomerase: Rotates the DNA to decrease torque (which would shred the helix) 3. Primase: Puts down a small RNA primer for DNA polymerase to bind to 4. DNA Polymerase III bonds nucleotides with unpaired bases 5. DNA polymerase I: replaces RNA primers with DNA 6. Ligase seals fragments together Final result = 2 exact copies of DNA * Each copy = 1 old strand and 1 new strand

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22 Replication Leading and Lagging Strand Nucleotides are added to the 3' end of the DNA strand replication can only occur in the 5' to 3' direction DNA is "anti-parallel": Both strands have opposite 5' to 3' orientations Leading strand made continuously Lagging strand made in smaller, discontinuous fragments (Okazaki fragments)

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24 Telomeres 1. Each round of replication shortens the 5' end of the lagging strand (by about bp) 2. If this continued indefinitely, chromosomes would get shorter and shorter after each replication. a. Information would start to be lost 3. TELOMERASE - enzyme responsible for adding telomeres at end of eukaryotic chromosomes a. short, repeating DNA sequence

25 Proofreading and Repair DNA polymerases proofread as bases added Errors: Pairing errors: 1 in 100,000 nucleotides Complete DNA: 1 in 10 billion nucleotides Mismatch repair: special enzymes fix incorrect pairings Nucleases cut damaged DNA DNA poly and ligase fill in gaps

26 Gene Expression: process by which DNA directs the synthesis of proteins (or RNAs) Old idea: one gene-one enzyme hypothesis Proposed by Beadle & Tatum mutant mold experiments Function of a gene = dictate production of specific enzyme Most accurate: one gene-one RNA molecule (which can be translated into a polypeptide)

27 Flow of genetic information Central Dogma: DNA RNA protein Transcription: DNA RNA Translation: RNA protein Ribosome = site of translation

28 Flow of Genetic Information in Prokaryotes vs. Eukaryotes

29 RNA (Ribonucleic Acid) and Transcription Structure of RNA # of strands Type of Sugar DNA RNA 2 Strands 1 Strand Deoxyribose sugar Ribose Sugar Nucleotide Base pairs A-T C-G A-Uracil C-G

30 RNA plays many roles in the cell 1. pre-mrna=precursor to mrna, newly transcribed and not edited 2. mrna= edited version; carries the code from DNA that specifies amino acids 3. trna= carries a specific amino acid to ribosome based on its anticodon to mrna codon 4. rrna= makes up 60% of the ribosome; site of protein synthesis 5. snrna=small nuclear RNA; part of a spliceosome; structural and catalytic roles 6. srprna= signal recognition particle that binds to signal peptides 7. RNAi= interference RNA; a regulatory molecule 8. mirna/sirna= micro/small interfering RNA; binds to mrna or DNA to block it, regulate gene expression, or cut it up 9. ribozyme= RNA that functions as an enzyme

31 3 types of RNA (All made in the nucleus) 1. Messenger RNA (mrna) a. Single straight strand b. Transmits DNA information c. Serves as template (pattern) for making proteins 2. Transfer RNA (trna) a. Single folded strand b. Complimentary bases pair up c. Involved in protein synthesis 3. Ribosomal RNA (rrna) a. Globular form b. Part of ribosome structure

32 Transcription making RNA from DNA 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)

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

35 Eukaryotes: TATA box = DNA sequence (TATAAAA) in promoter region upstream from transcription start site Transcription factors must recognize TATA box before RNA polymerase can bind to DNA promoter Transcription Factors + RNA Polymerase = 1. Initiation

36 RNA polymerase adds RNA nucleotides to the 3 end of the growing chain (A-U, G-C) As RNA polymerase moves, it untwists DNA, then rewinds it after mrna is made 2. Elongation

37 RNA polymerase transcribes a terminator sequence (prok) or polyadenylation signal sequence (euk), then mrna and polymerase detach. It is now called pre-mrna for eukaryotes. Prokaryotes = mrna ready for use 3. Termination

38 GENE

39 DNA mrna mrna Cytoplasm of cell Nucleus Transcription happens in the nucleus. An RNA copy of a gene is made. Then the mrna that has been made moves out of the nucleus into the cytoplasm Once in the cytoplasm, the mrna is used to make a protein

40 Processing mrna

41 Additions to pre-mrna: 5 cap (modified guanine) and 3 poly-a tail ( A s) are added Functions: 1. Export from nucleus 2. Protect mrna from enzyme degradation 3. Attach mrna to ribosomes in cytoplasm

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

43 RNA Splicing small nuclear ribonucleoproteins = snrnps snrnp = snrna + protein Pronounced snurps Recognize splice sites snrnps join with other proteins to form a spliceosome Spliceosomes catalyze the process of removing introns and joining exons Ribozyme = RNA acts as enzyme (catalytic role)

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

45 TRANSLATION - Protein Synthesis Proteins s to 1000 s of AA s per protein 1. linked by peptide bonds 2. Sequence of AA s determine structure and function of each protein

46 Codon a group of 3 bases on mrna different codons 2. Each codon codes for: a. 1 of the 20 amino acids b. Start or stop codons The code was cracked largely by Marshall Nirenberg Nobel Prize: 1968

47 Components of Translation 1. mrna = message 2. trna = interpreter 3. Ribosome = site of translation

48 trna Specific to each amino acid Transfer AA to ribosomes Anticodon: pairs with complementary mrna codon Base-pairing rules between 3 rd base of codon & anticodon are not as strict. This is called wobble.

49 Ribosomes Ribosome = rrna + proteins 2 subunits Active sites: A site: holds AA to be added P site: holds growing polypeptide chain E site: exit site for trna

50 Translation: 1. Initiation Small subunit binds to start codon (AUG) on mrna trna carrying Met attaches to P site Large subunit attaches

51 2. Elongation Codon recognition: trna anticodon matches codon in A site

52 2. Elongation Peptide bond formation: AA in A site forms bond with peptide in P site

53 2. Elongation Translocation: trna in A site moves to P site; trna in P site moves to E site (then exits)

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

55 Protein Folding During synthesis, polypeptide chain coils and folds spontaneously Chaperonin: protein that helps polypeptide fold correctly

56 Post-Translational Modifications Attach sugars, lipids, phosphate groups, etc. Remove amino acids from ends Cut into several pieces Subunits come together Insulin Production

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58 Polyribosomes: A single mrna can be translated by several ribosomes at the same time Translation in prokaryotes

59 Mutations changes in the genetic material of a cell Chromosomal: large-scale; always causes disorders or death (eg. nondisjunction, translocation, inversions, duplications, large deletions)

60 Mutagens: substances of forces that cause mutations in DNA

61 Types of Mutations Point Mutations: change single nucleotide pair of a gene 1. Substitution replace 1 with another Silent: same amino acid Missense: different amino acid Nonsense: stop codon, not amino acid 2. Frameshift (insertion/deletion/duplication) mrna read incorrectly; nonfunctional proteins

62 Substitution = Silent (no effect)

63 Substitution = Missense

64 Substitution = Nonsense (STOP)

65 Frameshift Mutation = Insertion/Deletion

66 Frameshift Mutation = Insertion/Deletion

67 3-Nucleotide-Pair Deletion/Insertion

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69 Sickle Cell Disease Symptoms Anemia Pain Frequent infections Delayed growth Stroke Pulmonary hypertension Organ damage Blindness Jaundice gallstones Life expectancy 42 in males 48 in females Caused by a genetic defect Carried by 5% of humans Carried by up to 25% in some regions of Africa

70 A Summary of Protein Synthesis Most current definition for a GENE: A region of DNA that can be expressed to produced a final product that is either a polypeptide or an RNA molecule