1. Chapter 15 Part I: Gene Regulation Part II: Mutations

2. Part I: Gene Regulation

3. Prokaryotic Gene Regulation Simpler organisms: simpler system. Easier to study. “Operon”: A region of the prokaryotic genome that encodes for several polypeptides all involved in a single metabolic pathway ACD E1 E2 E3 B

4. Concepts Repressible – Example: trp operon. – Encodes enzymes to synthesize the amino acid tryptophan. Usually this operon is “on”. However, if there is a lot of tryptophan, the operon will turn “off”. Generally associated with anabolic pathways (building stuff that isn’t there) Inducible – Example: lac operon – Encodes enyzmes that break down lactose into usable molecules (like glucose) – Usually off (the cell prefers glucose, not lactose). When there is little glucose, the cell turns this on so it can use an alternative energy supply. Generally associated with catabolic pathways (breaking stuff down)

5. Prokaryotic Gene Regulation E1, E2, E3, are all encoded along a single stretch of DNA, and all controlled by a single promoter region. Movie: the lac operon E1 E2E3 PromoterOperator

6. Additional Control To “fine tune” gene expression, all genes are controlled in more than one way (it’s not as simple as a repressor protein binding to an operator). Example: in addition to the concentration of lactose (which causes the repressor to fall off, remember), the concentration of glucose also helps control gene expression.

7. Additional Control High glucose – Low cAMP Low glucose – High cAMP – Binds to “CAP” (catabolite activating protein) – CAP/cAMP binds to CAP binding site next to the promoter. – Causes DNA to bend, favoring attachement of RNA polymerase High lactose – Repressor falls off, activating the lac operon Low lactose – Repressor stays attached to operator, preventing transcription

8. Eukaryotic Gene Expression Chromatin Structure – Histones, epigenetic inheritance, heterochromatin, euchromatin Transcriptional Control – Transcription factors – transposons Post-Transcriptional Control Translational Control Post-Translational Control

9. Part II: Mutations

10. Mutation Definition Used to be something that causes a change in phenotype. Now we know it’s a change in the DNA sequence of a gene.

11. Mutations Point Mutations Misssense Silent Nonsense Radical Conservative A change in the 3 rd base position of a codon. The protein sequence does not change. A mutation in a codon that changes the encoded amino acid. The new amino acid has strikingly different biochemical properties than the original The new amino acid has similar biochemical properties as the original A codon that normally encodes an amino acid is mutated to encode a “STOP” signal Frameshift A single base is inserted or deleted, resulting in the codons being read in a different reading frame. THE CAT ATE THE RAT THE ATA TET HER AT

12. 5’-UCUUAUUGUAGA-3’ UCU  UCA Ser  Ser

13. 5’-UCUUAUUGUAGA-3’ UCU  ACU Ser  Thr

16. 5’-UCUUAUUGUAGA-3’ UCU  UGU Ser  Cys

18. 5’-UCUUAUUGUAGA-3’ UCU  UGU Ser  Cys

19. 5’-UCUUAUUGUAGA-3’ AGA  UGA Arg  “STOP”

21. Mutations Effects – Protein function (remember hemoglobin) – Gene Regulation E.g. Mutate promoter region that affects RNA Polymerase binding. – Germ Cell Mutations (Evolutionarily Important) Mutations that occur in eggs and sperm. – Somatic Cell Mutations Somatic cells are body cells – the normal, diploid cells that are not involved in sexual reproduction. These mutations are NOT inherited. – Aging effects Mitochondrial mutation leads to aging Not necessarily… (good article)

23. 5’-ACGTACTATAGCAGCATGGATCGAATCGATACACT-3’ TATA Box is a promoter sequence that the protein TBP binds. TBP (TATA Binding Protein) recruits RNA Polymerase to the promoter to turn it “on”. Notice the start site. If “TATA” gets mutated, TBP can’t bind. RNA Polymerase is no longer recruited. The gene is shut down.

24. Mutations Causes – DNA replication errors Prokaryotes have high error rates; eukaryotes have very low (1 in a billion). But they DO happen.

25. Mutations Causes – Radiation Energy from subatomic particles is absorbed by organic molecules. This causes bonds to break. The high energy biproducts form new bonds. These new bonds are abnormal. If this happens within DNA, it leads to changes in the genetic code.

26. DNA Damage To Nuclear Test Vets Prompts Call For Study Of Children (5/16/2007)

27. Mutations Causes – Chemicals From the National Cancer InstituteNational Cancer Institute Cigarette smoke contains about 4,000 chemical agents, including over 60 carcinogens (8). In addition, many of these substances, such as carbon monoxide, tar, arsenic, and lead, are poisonous and toxic to the human body. Nicotine is a drug that is naturally present in the tobacco plant and is primarily responsible for a person’s addiction to tobacco products, including cigarettes. During smoking, nicotine is absorbed quickly into the bloodstream and travels to the brain in a matter of seconds. Nicotine causes addiction to cigarettes and other tobacco products that is similar to the addiction produced by using heroin and cocaine (9).arsenic toxicdrugaddiction Nicotine itself inhibits apoptosis.

28. double strand DNA damage Single strand DNA damage

29. Like a watch wrapped around a wrist, a special enzyme encircles the DNA double helix. Using a combination of imaging techniques, researchers have captured snapshots of the enzyme, DNA ligase, joining together a broken strand of DNA. Millions of DNA breaks occur during the normal course of a cell's life. Without molecules that can connect the pieces, cells can malfunction, die, or become cancerous. Courtesy of Tom Ellenberger, Washington University School of Medicine in St. Louis

30. Mutations Repair – Role of DNA polymerase 3'->5' exonuclease activityexonuclease One example: (from same ref as below): Oxidation of G to form oxoG produces a subtle structural transformation – This results in deleterious mutations because DNA polymerases misread oxoG as a thymine (T) base during replication. – repair enzymes (ref)ref DNA-repair enzymes have the ability to search through vast tracts of DNA to find subtle anomalies in the structure The information content of the DNA double helix is preserved by a crew of DNA-repair enzymes that defend the genome from the harmful effects of DNA damage

31. Note the difference between

32. Quick! – Lampbrush chromosomes Lampbrush chromosomes – Polytene chromosomes Polytene chromosomes

33. Polytene Chromosomes Polytene chromosomes form when multiple rounds of replication produce chromatids that remain synapsed together in a haploid number of chromosomeschromosomeschromatidshaploid