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Tutorial - DNA Watson & Crick Griffith’s Experiments (Streptococcus pneumoniae) Experiment Results Conclusion 1 R-Strain bacteria S-Strain bacteria Mouse lived Mouse died Nonvirulent strain of bacteria Virulent/deadly strain 2 Heat-killed S-Strain Mouse lived Destroyed deadly bacteria 3 Heat-killed S-Strain + R-Strain Mouse died Some how the killed bacteria was able to pass “something” to the R-strain That Transformed it to become deadly Avery’s Experiments (enzymes & bacteria) Experiments Results Conclusion Enzymes to break down proteins, carbohydrates, lipids, RNA, & finally DNA Deadly in spite of all enzymes except one that broke apart DNA DNA is the transforming molecule that made Griffith’s R-Strain bacteria turn into an S-Strain bacteria Hershey & Chase Experiments (bacteria & a virus called bacteriophage) Experiments Results Conclusion Radioactive Protein Radioactive DNA New phages = no radioactivity New phages = radioactive Protein does not make new phages DNA makes new phages DNA Structure: 1. Nucleosides 2. Nucleotides 3. Bases Purines Adenine, Guanine Pyrimidines Cytosine, Thymine 4. Chargaff’s Rule 5. Franklin/Wilkins Watson/Crick 1 hexagon ring + 1 pentagon ring Anti-Parallel 5’ – 3’ 5’ 3’ 1 hexagon ring A bonds to T C bonds to G 3’ 5’ Conclusion-DNA is a helical structure With distinctive regularities. nucleotide PO4 How would your replicate this DNA molecule? N base 5 CH2 O 4 deoxyribose 3 OH 2 1 Which is the leading strand? Lagging strand? Which one has Okasaki Fragments? What enzymes are involved? What type of bonds are formed? What is the end result? Replication fork DNA polymerase I DNA polymerase III lagging strand Okazaki fragments 5’ 3’ primase ligase 5’ SSB 3’ DNA polymerase III 5’ 3’ leading strand direction of replication 3’ 5’ helicase Replication enzymes Helicase - unzips DNA single-stranded binding proteins - controls the unzipping of DNA DNA polymerase III - main DNA building enzyme Primase - lays down RNA primer on lagging strand DNA polymerase I - editing, repair & primer removal ** Ligase - “glues” Okazaki fragments together on lagging strand Telomeres Expendable, non-coding sequences at ends of DNA short sequence of bases repeated 1000s times TTAGGG in humans Telomerase enzyme in certain cells enzyme extends telomeres prevalent in cancers Ends of chromosomes are eroded with each replication an issue in aging? telomeres protect the ends of chromosomes Genetic Material Prokaryotic DNA • Circular in shape • In the cell’s cytoplasm • Not wrapped around proteins • Fewer average bp’s (base pairs) • No introns Eukaryotic DNA • Linear in shape • In the cell’s nucleus • Wrapped around proteins • More bp’s (base pairs) • Introns and exons What would happen if you put a eukaryotic DNA into a prokaryote? Regulating Gene Expression DNA is tightly wound around histone proteins, making DNA inaccessible to enzymes that would code for the genetic information Acetyl groups attach to the histones Causing the tight compaction to unravel, now allowing DNA to be susceptible to activation (replication or transcription) A methyl group (CH3) can be attached to a cytosine base on DNA, as shown here. When a methyl group is attached to a base, the base cannot be accessed to build nucleotides Implications: What effect would that have on the gene’s expression? Ribosomes Prokaryotic ribosomes • 70S (smaller) • Synthesized and assembled in the cytoplasm • Simultaneous transcription and translation • Translation begins with f-met • Sensitive to antibiotics Eukaryotic ribosomes • 80S (larger) • Synthesized in the nucleolus • Assembled in the cytoplasm (free) or (attached) on the Rough Endoplasmic Reticulum • Transcription then translation • Translation begins with met Both • Translation is powered by GTP (guanosine triphosphate) • Terminate translation with a stop codon & release factor proteins The “Central Dogma” flow of gene tic information within a cell transcription DNA replication RNA translation protein DNA - RNA - Protein All RNA’s (mRNA, rRNA, tRNA) are transcribed (made) in the nucleus Transcription - RNA Where is 5’ and 3’? the making of mRNA from a DNA template Post-transcriptional processing Primary transcript eukaryotic mRNA needs work after transcription Protect mRNA from RNase enzymes in cytoplasm add 5' cap mRNA 5' cap PPP add polyA tail 5' GCH Edit out introns 3' A 3 intron = noncoding (inbetween) sequence eukaryotic DNA exon = coding (expressed) sequence primary mRNA transcript mature mRNA transcript pre-mRNA spliced mRNA Transcription in Prokaryotes Initiation RNA polymerase binds to promoter sequence on DNA Role of promoter 1. Where to start reading = starting point 2. Which strand to read = template strand 3. Direction on DNA = always reads DNA 3'5’ = transcribes DNA 5’3’ What do prokarotic mRNA lack in comparison to eukaryotic mRNA’s? Ribosomes – made of rRNA and protein P site (peptidyl-tRNA site) holds tRNA carrying growing polypeptide chain A site (aminoacyl-tRNA site) holds tRNA carrying next amino acid to be added to chain E site (exit site) empty tRNA leaves ribosome from exit site tRNA structure “Clover leaf” structure anticodon on “clover leaf” end amino acid attached on 3' end Building a polypeptide Initiation brings together mRNA, ribosome subunits, proteins & initiator tRNA Elongation Termination RNA polymerase DNA Can you tell the story? amino acids exon intron tRNA pre-mRNA 5' cap mature mRNA aminoacyl tRNA synthetase polyA tail large subunit polypeptide ribosome 5' small subunit tRNA E P A codon 3' Put it all together… Lactose digestion in E.coli begins with its hydrolysis by the enzyme ß-galactosidase. The gene encoding ß-galactosidase, lacZ, is part of a coordinately regulated operon containing other genes required for lactose utilization. Which of the following figures correctly depicts the interactions at the lac operon when lactose is NOT being utilized? (The legend below defines the shapes of the molecules illustrated in the options.) Lac Operon What’s it sound like it involves? Lac = Lactose; Operon = Operates when it’s On Which of the following figures correctly depicts the interactions at the lac operon when lactose is NOT being utilized? Implications to Genetically modified plants: a. Pest resistance? b. Herbicide resistance?