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MCB 317 Genetics and Genomics

MCB 317 Genetics and Genomics

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MCB 317 Genetics and Genomics

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  1. MCB 317Genetics and Genomics Topic 9 Overview of Eukaryotic Gene Expression

  2. Gene Regulation in Eukaryotes

  3. Readings Chromatin: Hartwell Chapter 12, pages 405-410 Heterochromatin: Hartwell Chapter 12, section 12.3

  4. Gene Expression v. Transcription Concept: Every step in a biological process is a potential site of regulation

  5. Outline • Txn in Prokaryotes • Overview of Txn in Eukaryotes • DNA Binding Proteins (“Txn Factors”) • Chromatin • Knowledge / Facts / Language • Knowing HOW we know what we know • Asking new questions & discovering answers

  6. Expectations and Review • Prokaryotes: Basic process and nomenclature • Process of txn • Start and stop signals for txn • Gene orientation • One RNAP

  7. Expectations and Review Nomenclature: ORF, promoter, codon, Start/stop codons, mRNA, untranslatedregion, tRNA, consensus sequence, homolog, coding and noncoding strands, activator (proteins), repressor, etc…

  8. Prokaryotes Consensus sequence

  9. Lodish 11-9 TATA(A/T)A(A/T)(A/G) Why consensus and not exact sequence? How Does RNAP “find” its Promoter and Initiate Txn?

  10. Consensus sequences provide for binding to specific DNA sites over a range of affinities Concept: Biological Reactions are often Optimized, not Maximized

  11. Synthesis/polymerization is in the 5’ to 3’ direction

  12. Coding v. non-coding strand, Directionality Coding looks like mRNA Non-coding can base-pair With mRNA

  13. Promoters are Directional

  14. Activators

  15. Repressors

  16. Concept:Turning Genes ON and OFF ON -> Activated OFF -> Repressed OFF -> Not Activated In General: Repressors Win

  17. Distinguishing: Activators from Repressors Positive Regulators from Negative Regulators Key: What is the role of the active form of the protein

  18. Regulation: Activation/Repression in Response to Particular Conditions

  19. Repressors Activators and Repressors vs. Inducers

  20. Outline • Txn in Prokaryotes (Review) • Overview of Txn in Eukaryotes • Chromatin

  21. Thinking About Prokaryotic v. Eukaryotic Txn 1. Dynamic Range of Regulation: Prokaryotes v. Eukaryotes A. E.coli ON:OFF = 200-1000:1 max. Most “OFF” genes about 100 x below ON B. Most Eukaryotes ON:OFF = 108:1

  22. Thinking About Eukaryotic Txn 2. Genome size How do Regulatory proteins find their targets in the face of 1000 fold increase in “non-specific” DNA? 3. Chromatin and Higher order DNA packaging Concept: Euk genomes are more complex; therefore, the txn machinery is more complex

  23. Three Eukaryotic RNAPs RNAP I -> rRNA genes RNAP II -> protein coding RNAP III ->tRNA, 5S rRNA, other small RNAs Basic Machinery Conserved Yeast -> Humans

  24. DNA Sequence Elements and DNA Binding Proteins “trans” factors = proteins or complexes “cis” sequence elements DNA sequence elements that regulate transcription typically bind specific regulatory proteins or protein complexes

  25. Eukaryotes: Tighter regulation Larger range of regulation Larger genome Multicellular Chromatin More Complex Regulation

  26. Enhancers, Activators Promoter, Basal Factors = General Factors Language Caution: Genetic Activator vs. a Txn Activator protein = Activator

  27. Watson 9-6 and 9-8 Enhancers= short regions (typically ~ 200 bp) of densely packed consensus elements Some elements found in both promoters and enhancers

  28. Eukaryotic txn = large protein complexes Lodish 11-35

  29. Complex of complexes ~ 100 proteins Lodish 11-36

  30. Txn in the face of chromatin and higher order packing Lodish 11-37

  31. Enhancers act independently and cumulatively Reporter Genes

  32. Reporter Genes

  33. E1 E1 Pr Coding Region E1 E1 Pr Reporter Cod. Reg. Reporter Genes Reporter Genes typically code for easily visualized protiens: lacZ = enzyme: colorless precursor -> blue product GFP = Green flourescent protein (Jellyfish)

  34. Reporter Genes

  35. For Sub-cellular Localization For Txn Pattern: E1 Pr GFP For Expression Pattern (and subcellular local): txn and translation E1 Pr Coding Region GFP

  36. For Expression Pattern: txn and translation E1 Pr Myo2 GFP

  37. Sub-cellular localization of splicing factors Splicing Factor-GFP fusion

  38. Biochemistry 1 Protein Gene (Organism 2) 2 6 9 5 4 Gene Ab 7 12 3 8 Expression Pattern Mutant Gene 10 Mutant Organism 11 Genetics

  39. Molecular Genetics Summary • Column Chromatograpy (ion exchange, gel filtration) • A. Make Polyclonal Ab; B. Make Monoclonal Ab • Western blot, in situimmuno-fluorescence (subcellular, tissue) • Screen expression library (with an Ab) • Screen library with degenerate probe • Protein expression (E. coli) • A. Differential hybridization • A. Northern blot, in situ hybridization, GFP reporter, GFP Fusion • A. low stringency hybridization; B. computer search/clone by phone; C. computer search PCR • Clone by complementation (yeast, E. coli) • A. Genetic screen; B. genetic selection • RNAi “knockdown”

  40. DNA Sequence Elements and DNA Binding Proteins “trans” factors = proteins or complexes “cis” sequence elements DNA sequence elements that regulate transcription typically bind specific regulatory proteins or protein complexes

  41. Regulation: Activation/Repression in Response to Particular Conditions

  42. DNA elements (sequence elements) act by binding proteins The proteins do the work