2. Tutorial - DNA Watson & Crick

3. Griffith’s Experiments (Streptococcus pneumoniae) ExperimentResultsConclusion 1R-Strain bacteria S-Strain bacteria 2Heat-killed S-Strain 3 + R-Strain Avery’s Experiments (enzymes & bacteria) Nonvirulent strain of bacteria Destroyed deadly bacteria Some how the killed bacteria was able to pass “something” to the R-strain That Transformed it to become deadly ExperimentsResultsConclusion Enzymes to break down proteins, carbohydrates, lipids, RNA, & finally 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) ExperimentsResultsConclusion Radioactive Protein Radioactive DNA Protein does not make new phages DNA makes new phages Mouse lived Mouse died Deadly in spite of all enzymes except one that broke apart DNA New phages = no radioactivity Mouse died Virulent/deadly strain New phages = radioactive

4. Anti-Parallel 5’ – 3’ DNA Structure: 1. Nucleosides 2. Nucleotides 3. Bases 1 hexagon ring + 1 pentagon ring 1 hexagon ring 4. Chargaff’s Rule Purines Adenine, Guanine Pyrimidines Cytosine, Thymine 5’5’ 5’5’ 3’3’ 3’3’ A bonds to T C bonds to G 5. Franklin/Wilkins Watson/Crick Conclusion-DNA is a helical structure With distinctive regularities.

5. OH CH 2 O 4 5 3 2 1 PO 4 N base deoxyribose nucleotide How would your replicate this DNA molecule? 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?

6. Replication fork 3’3’ 5’5’ 3’3’ 5’5’ 5’5’ 3’3’ leading strand Okazaki fragments lagging strand 3’3’5’5’ DNA polymerase III ligase helicase direction of replication primase DNA polymerase III SSB DNA polymerase I

8. 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

9. 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

10. Genetic Material Prokaryotic DNA Eukaryotic DNA Circular in shape In the cell’s cytoplasm Not wrapped around proteins Fewer average bp’s (base pairs) No introns 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?

11. 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) Regulating Gene Expression A methyl group (CH 3 ) 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?

12. Ribosomes Prokaryotic ribosomes Eukaryotic ribosomes 70S (smaller) Synthesized and assembled in the cytoplasm Simultaneous transcription and translation Translation begins with f-met Sensitive to antibiotics 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

13. proteinRNA The “Central Dogma” DNA transcriptiontranslation replication  flow of gene  tic information within a cell

14. DNA - RNA - Protein All RNA’s (mRNA, rRNA, tRNA) are transcribed (made) in the nucleus

15. Transcription - the making of mRNA from a DNA template RNA Where is 5’ and 3’?

16. A A A A A 3' poly-A tail CH 3 mRNA 5' 5' cap 3' G PPP Post-transcriptional processing  Primary transcript  eukaryotic mRNA needs work after transcription  Protect mRNA  from RNase enzymes in cytoplasm  add 5' cap  add polyA tail  Edit out introns eukaryotic DNA exon = coding (expressed) sequence intron = noncoding (inbetween) sequence primary mRNA transcript mature mRNA transcript pre-mRNA spliced mRNA 50-250 A’s

17. 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’ Transcription in Prokaryotes  Initiation  RNA polymerase binds to promoter sequence on DNA What do prokarotic mRNA lack in comparison to eukaryotic mRNA’s?

18. 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

19. tRNA structure  “Clover leaf” structure  anticodon on “clover leaf” end  amino acid attached on 3' end

20. Building a polypeptide  Initiation  brings together mRNA, ribosome subunits, proteins & initiator tRNA  Elongation  Termination

21. Can you tell the story? DNA pre-mRNA ribosome tRNA amino acids polypeptide mature mRNA 5' cap polyA tail large subunit small subunit aminoacyl tRNA synthetase EPA 5' 3' RNA polymerase exon intron tRNA codon

22. Put it all together…

23. 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.)

24. 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?

25. Implications to Genetically modified plants: a. Pest resistance? b. Herbicide resistance?