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Regulation of Transcription (Ch13)

Regulation of Transcription (Ch13). Genetic Analysis of Regulation of Transcription Identification and characterization of cis-acting and trans-acting regulators Detailed analysis of the E. coli lac operon General information on eukaryotic cis- and trans-acting elements

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Regulation of Transcription (Ch13)

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  1. Regulation of Transcription (Ch13) • Genetic Analysis of Regulation of Transcription • Identification and characterization of cis-acting and trans-acting regulators • Detailed analysis of the E. coli lac operon • General information on eukaryotic cis- and trans-acting elements • Gal4 illustrates the domain structure of trans-acting regulators • Gal4 system developed into the famous “two-hybrid assay” for interacting proteins (end of Chapter 9) • Paradigms for coordinate control of transcription for multiple genes • Operons in prokaryotes • Multiple Sigma factors in prokaryotes • Multiple RNA polymerases in eukaryotes • Multiple genes under control of one trans-acting factor • Other Examples of transcriptional regulatory mechanisms • Gene rearrangements • Epigenetic regulation

  2. Regulation of Genes involved in Carbon Source Metabolisms • Glucose • Lactose • Arabinose • Sucrose • Ethanol • Others…

  3. Diauxic Growth: bacteria use one carbon source at a time lactose glucose

  4. Lactose utilization genes: The Lac Operon

  5. Genetic Analysis of Lac Operon RegulationlacZ and lacO mutants

  6. Genetic Analysis of Lac Operon RegulationlacI mutants

  7. Genetic Analysis of Lac Operon RegulationlacI super repressor mutants

  8. Promoter mutants

  9. Z Y Gene - + - + Inducer - - - - #1 Result + #2 Result - + - -

  10. Regulation of Transcription (Ch13) • Genetic Analysis of Regulation of Transcription • Identification and characterization of cis-acting and trans-acting regulators • Detailed analysis of the E. coli lac operon • General information on eukaryotic cis- and trans-acting elements • Gal4 illustrates the domain structure of trans-acting regulators • Gal4 system developed into the famous “two-hybrid assay” for interacting proteins (end of Chapter 9) • Paradigms for coordinate control of transcription for multiple genes • Operons in prokaryotes • Multiple Sigma factors in prokaryotes • Multiple RNA polymerases in eukaryotes • Multiple genes under control of one trans-acting factor • Other Examples of transcriptional regulatory mechanisms • Gene rearrangements • Epigenetic regulation

  11. Genetic analysis of lac operon regulation using partial diploids: 1. Draw both operons 2. Note of cis-acting mutations - what is phenotype for each operon 3. Note trans-acting mutations - does this change result in (2) above? Z Y Gene - + - + Inducer - - + + #3 Result - - + + #4 Result

  12. Sequence-specific DNA Binding Proteins: example of lacI and CAP sites LacI LacI

  13. Characterization of cis-acting sequences by DNA foot printing

  14. Regulation of Transcription in Eukaryotes • Cis-acting elements • promoter • enhancers (positive acting) • silencers (negative acting) • trans-acting factors • general (or basal) transcription factors • activators • coactivators (or adaptors) • repressors

  15. Eukaryotic promoter elements

  16. Role of eukaryotic trans-acting transcription factors

  17. Genetic Analysis of Regulation of Transcription in Eukaryotes • Isolate and analyze mutations in cis and trans-acting element • Use recombinant DNA and transgenic cells/animals • construct gene fusions in vitro • transcriptional fusions • translational fusions • fusions to LacZ coding sequence • transform gene fusions into cells/organisms • determine relative levels of Beta-Galactosidase activity

  18. Paradigms for coordinate control of transcription for multiple genes Operons in prokaryotes Multiple Sigma factors in prokaryotes Multiple RNA polymerases in eukaryotes Multiple genes under control of one trans-acting factor

  19. Epigenetic Control: Regulation of gene expression by DNA or chromatin modification

  20. Epigenetic Control: Regulation of gene expression by DNA or chromatin modification

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