Regulation of Gene Expression Ch. 16.1-16.2;16.4-16.5

1. Regulation of Gene ExpressionCh. 16.1-16.2;16.4-16.5

2. 1 Embryo 200 Cell Types • From a single embryo, 200 types of cells can be produced (differentiation) • Diversity comes from genes being turned off • Expression of the genes lead to specialization of the cell • Transcriptional regulation controlling the expression of genes • post-transcriptional effect mRNA • Translational protein translation • Post-translational life span/activity of protein

3. Regulation in Prokaryotes • Adjust biochemistry quickly as environment changes • Jacob and Monod extensive studies into the effects of lactose on expression of lactase genes • Operon regulatory sequence in DNA for a specific gene(s) + the genes • Regulatory proteins bind to operons to promote or inhibit the transcription of transcription unit (single mRNA coded in the operon)

4. Regulation of an Operon • Operator section at the start of the operon • Activator protein attaches to operator to promote expression • Repressor protein attaches to operator to inhibit expression • Gene coding for regulatory proteins (activators/repressors) are called regulatory genes • Non-regulating proteins come from structural genes

5. The lac Operon • 3 genes: • lacZ codes for β-galactosidase; breaks lactose into glucose + glactose • lacYcodes for permease; actively transports lactose into the cell • lacA codes for transacetylase; We don’t know what it does • Negatively regulated • Regulator gene lacI codes for Lac repressor • Limits lac expression when lactose is absent (normal) • When lactose is added, it is made into allolactose(inducer for lac operon) • Inhibits lac repressor by binding to it

6. Lac Operon Part II; Positive Regulation • Lac operon is repressed in the presence of lactose if glucose is also added. Why? • Glucose is a better source of energy • Converting lactose into usable sugars (glucose) requires energy • CAP (catabolite activator protein) activator synthesized in an inactive form; activated by cAMP (produced when glucose is absent) • Active form binds to CAP site at the lac operon promoter allowing RNA Poly to attach • If we add glucose, cAMP levels drop so CAP is deactivated and RNA Poly can bind to the DNA

7. trp Operon and Protein Synthesis • Some proteins, like tryptophan, must be synthesized when not present to be absorbed • trp Operon codes enzymes needed to make tryptophan; regulated by trpR (repressor) that is normally inactive; trp operon used to make tryptophan • When tryptophan levels are high, the repressor is active and trp operon is blocked (repressible operon) • Tryptophan is a corepressor; activates repressor

8. Regulation in Eukaryotes • Eukaryotes do not have operons; regulatory gene are spread across the genome (side effect of variation) • Eukaryotes use all forms of gene regulation: • Transcriptional Regulation • Post-transcriptional regulation • Translational regulation • Post-translational regulation

9. Transcriptional Regulation • Promoter region of DNA upstream (~25bp) from the transcription unit • TATA Box 7-bp sequence 5’-TATAAAA-3’ • TFs (transcription factors) recognize TATA and bind to it; then RNA Poly II can bind • Further upstream are the regulator sequences (promoter proximal elements) in the promoter proximal region • Regulatory proteins bind here to enhance or repress transcription

10. Activators and Transcription • RNA Poly II + TFs transcription initiation complex; not that efficient • Activators proteins that help the complex attach and start translation • Activators can be specific (one cell type for one gene) or general (multiple genes in all cell types) which are also called Housekeeping genes • Enhancer regions on the DNA can increase transcription rate by interacting with activators (act as coactivators) by bending DNA into a loop

11. Motifs in DNA Binding Proteins • Domains structures in a protein made from the combination of secondary folding options (helix, sheet, coil) • Ex. Helix-helix-coil-helix • Motif specialized domains conserved in different types of proteins • DNA interacting Motifs: • Helix-turn-helix DNA binding region of protein • Zinc Finger finger shape with zinc ion; bind to DNA grooves • Leucine zipper dimers held together by hydrophobic regions; bind to major groove of DNA

12. Combinational Gene Regulation • Regulation of most genes in more complex than just activation or repression • Genes can have multiple activators and repressors • These regulation points between different genes overlap and follow the stronger influence • Gene A is regulated by enhancer regions 1, 2 and 3; Gene B is regulated by enhancer 2, 3, and 4 • Activators on 2 and 3 will produce A and B proteins • Repressors on 3, and 4 will limit B protein a great deal and A proteins a little bit

13. Coordinated Regulation • Proteins can be regulated in complex organisms across many types of tissues through chemical signals (hormones) • Steroid Hormone Response Element region in gene that hormone-receptor complex binds to • Allows regulation in several cell types very quickly

14. Methylation of DNA • DNA methylation adding methyl (-CH3) to cytosine bases • Turn off gene (silencing) by blocking access to promoter region • Epigenetics change in gene expression but no change in the DNA itself • Hemoglobin turned off in all other cell types this way • Genomic Imprinting silencing of one of two alleles during development • Methylated allele is not expressed

15. Chromatin Structure • Histones can block access to DNA and thus regulate it • Chromatin remodeling changing its structure • Nucleosome remodeling complex moves histones along DNA or reshapes them to open a region • Adding Acetyl Groups (CH3CO-) weakens the interactions between the histones and DNA • Methylation of Histones marks histones wrapped with deactivated DNA

16. Gene Regulation in Development • Gene regulation is most important during early development; determine the cell-types and physiology of the organism • Regulation sensitive to both time (must all happen in the right order and within a certain window) and place (location in embryo determines location in body) • Understanding comes from our model organisms: • Fruit fly, nematode worm, zebrafish, and house mouse

17. From Zygote to Fetus • After fertilization, a zygote develops into a fetus through several mechanisms • Mitosis need lots of cells • Movement of cells cells need to form the right shape • Induction cell of a certain type needs neighboring cells to respond to get a result • Determination totipotent cells becomes specific cell types • Differentiation cell types become finalized so tissue and systems can be made

18. Hold Up Mr. Nucleus…Cytoplasm has something to say… • Not all regulation of a zygote comes from the nucleus • Zygote’s cytoplasm is from the egg used at fertilization • Cytoplasmic determinants • mRNA strands and proteins in cytoplasm of egg also regulate the zygote • Not reproduced during cell divisions; First divisions of zygote separate determinants asymmetrically so each daughter as an uncontrolled amount • Only really take effect during the first few divisions but can last till tissues form • Inherited only on the maternal side

19. Induction • Major step in the process of determination • Signal molecules from very specific cells (inducers) sent to receptor cells • Two methods: • Signal released and travels short distances to receptors • Cell-to-Cell contact between proteins in the membranes of inducers and receptors

20. Differentiation • Determination narrows the type of cells possible and differentiation limits to one cell type • Genes required for cell type are left on while other genes are “turned off” • Master regulatory genes promote the transcription of proteins needed to specialize the cell • myoD master gene regulates MyoD transcription factors which promotes skeletal muscle proteins

21. Physical Position and Regulation • Pattern formation arrangement of organs in the body • Discovered studying the effects of mutations on the embryogenesis of fruit flies • Particular genes control the body plan for all complex organism • Steps required: • Determine front, back, head, and tail (ventral, dorsal, anterior, and posterior) of embryo • Divided zygote into segments • Use segments to map out body plan

22. Maternal-Effect Genes • Expressed when egg is produced by the mother; mRNAs made from the bicoid gene • Control the anterior-to-posterior polarity of the egg (front to back) • Bicoid protein is produced and the highest conc. marks the anterior (head) and drops as move along to the posterior (butt) which has the lowest conc.

23. Segmentation Genes • 24 genes divide embryo into regions • 3 Types: • Gap Genes form segments along A-P axis; broad regions • Pair-rule Genes divide broad regions with units of two segments each • Segment polarity Genes sets the boundaries for each segment; each segments needs an A-P axis

24. Homeotic Genes • Genes specify which segment becomes what; where are the legs, eyes, wings, etc… • Hox genes • 8 Hox genes in fruit flies • Actually occur in order on chromosome (AP) • Found in all animals and is highly conserved • Homeo-Box region in all homeotic genes that codes for its specific homeodomain(TF for its protein)

25. Genes and Cancer • 2 types of Cancer • Familial Cancer inherited; common with breast, colon, and testicular cancers • Sporadic Cancer occur randomly; more common form; can happen from viruses altering DNA • All cancer is a multi-step process; need several key mutations • 3 Classes of Genes effect cancer frequency: • Proto-oncogens • Tumor suppressor genes • microRNA genes • (not covering this)

26. Proto-Oncogenes • Genes that stimulate cell division in regular healthy cells • Code for growth factors, signal receptors, transduction components, and TFs • When mutated, they can become overactive oncogens • Only one allele needs mutated to take effect • Mutation in the promoter • Mutation in the transcription unit • Translocation moves gene to a more active promoter or enhancer • Virus adds genes that activate or enhance a gene

27. Tumor Suppressor Genes • Code for proteins that inhibit cell division • Keep Proto-oncogenes repressed • TP53 codes for p53 that inhibits CDKs used to pass the G1/S checkpoint • If mutated, p53 can’t inhibit division • p53 mutations are in 50% of all cancers • Both alleles must be inactive for a tumor suppressor gene to lose function

28. Homework • Suggested Homework: • Test Your Knowledge Ch. 16 • Actual Homework: • Discuss the Concepts #1 • Interpret the Data Ch. 16 • Design the Experiment Ch. 16

29. Assignments for Next Week • PPT Presentations on Ch. 18: • Groups of 3; 12-15 mins long • Topics: • DNA Cloning and Building DNA Libraries • Gel Electrophoresis, Southern Blot, Northern Blot, and Western Blot • DNA Cloning and Bacteria Transformation for Protein Synthesis • BLAST Program and How it is Used • Papers on Ch. 19: • 3 page paper discussing the following: • Darwin’s Journey • Data and Experiments by Darwin • World Reaction to Darwin’s Theories • Basic Principles of Evolution • DO NOT answer these section by section. These are the BIG IDEAS you paper must discuss. It should be a summary of Darwin’s life and impact on Biology