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Regulation of Prokaryotic and Eukaryotic Gene Expression

Regulation of Prokaryotic and Eukaryotic Gene Expression. A bacterium can tune its metabolism to the changing environment and food sources This metabolic control occurs on two levels: Adjusting activity of metabolic enzymes Regulating genes that encode metabolic enzymes.

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Regulation of Prokaryotic and Eukaryotic Gene Expression

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  1. Regulation of Prokaryotic and Eukaryotic Gene Expression

  2. A bacterium can tune its metabolism to the changing environment and food sources • This metabolic control occurs on two levels: • Adjusting activity of metabolic enzymes • Regulating genes that encode metabolic enzymes

  3. Regulation of enzyme production Regulation of enzyme activity Precursor Feedback inhibition Enzyme 1 Gene 1 LE 18-20 Gene 2 Enzyme 2 Regulation of gene expression Gene 3 Enzyme 3 Enzyme 4 Gene 4 Gene 5 Enzyme 5 Tryptophan

  4. Operons: The Basic Concept • In bacteria, genes are often clustered into operons, composed of • An operator, an “on-off” switch • A promoter • Genes for metabolic enzymes

  5. trp operon Promoter Promoter LE 18-21a Genes of operon DNA trpB trpA trpE trpC trpD trpR Operator Stop codon RNA polymerase Regulatory gene Start codon 3¢ mRNA 5¢ mRNA 5¢ D B E C A Inactive repressor Protein Polypeptides that make up enzymes for tryptophan synthesis Tryptophan absent, repressor inactive, operon on

  6. DNA LE 18-21b_1 mRNA Active repressor Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off

  7. DNA No RNA made LE 18-21b_2 mRNA Active repressor Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off

  8. Two Types of Negative Gene Regulation • A repressible operon • Is usually on • binding of a repressor to the operator shuts off transcription • The trp operon is a repressible operon • An inducible operon • Is one that is usually off • a molecule called an inducer inactivates the repressor and turns on transcription • the lac operon is an inducible operon, which contains genes coding for enzymes in hydrolysis and metabolism of lactose

  9. Promoter Regulatory gene Operator lacl lacZ DNA LE 18-22a No RNA made 3¢ mRNA RNA polymerase 5¢ Active repressor Protein Lactose absent, repressor active, operon off

  10. lac operon DNA lacl lacY lacA lacZ LE 18-22b RNA polymerase 3¢ mRNA mRNA 5¢ 5¢ Transacetylase Permease -Galactosidase Protein Inactive repressor Allolactose (inducer) Lactose present, repressor inactive, operon on

  11. Inducible enzymes usually function in catabolic (“breakdown”) pathways • Explain to a neighbor why this makes sense. • Repressible enzymes usually function in anabolic (“synthesis”) pathways • Explain to a neighbor why this makes sense. • Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor

  12. Positive Gene Regulation • Some operons are also subject to positive control through a stimulatory activator protein, such as catabolite activator protein (CAP) • When glucose (a preferred food source of E. coli ) is scarce, the lac operon is activated by the binding of CAP • When glucose levels increase, CAP detaches from the lac operon, turning it off

  13. Promoter DNA lacl lacZ LE 18-23a RNA polymerase can bind and transcribe Operator CAP-binding site Active CAP cAMP Inactive lac repressor Inactive CAP Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized

  14. Promoter DNA lacl lacZ LE 18-23b CAP-binding site Operator RNA polymerase can’t bind Inactive CAP Inactive lac repressor Lactose present, glucose present (cAMP level low): little lac mRNA synthesized

  15. Eukaryotic Gene Regulation

  16. Every cell in a multi-cellular eukaryote does not express all its genes, all the time (usually only 3-5%) • Long-term control of gene expression in tissue = differentiation • How to prevent expression? • Regulation at transcription • Regulation after transcription

  17. Chromatin Regulation • Chromatin remodeling allows transcription • Chromatin = DNA + proteins • Chromatin coiled around histones = nucleosomes • Allows DNA to be packed into nucleus, but also physically regulates expression by making regions ‘available’ or not

  18. Chromatin regulation can be small-scale (gene) or large scale (chromosome) • Non-expressed = heterochromatin (condensed) • Expressed = euchromatin (relaxed)

  19. Changes to Chromatin (DNA) • Methylation • Methylating (adding methyl groups) to DNA bases, keeping them “tight” and “closed” – inaccessible to transcription. • Histone Acetylation • “Acetylating” histones (adding acetyl groups) promotes loose chromatin and permits transcription

  20. Transcription Regulation • What we know from prokaryotes: • Several related genes can be transcribed together (ie. lac operon) • Need RNA Polymerase to recognize a promoter region • Why eukaryotes are different: • Genes are nearly always transcribed individually • 3 RNA Polymerases occur, requiring multiple proteins to initiate transcription

  21. Transcription Regulation Con’t • Typical prokaryotic promoter: recognition sequence + TATA box -> RNA Polymerase -> transcription • Typical eukaryotic promoter: recognition sequence + TATA box + transcription factors -> RNA Polymerase -> transcription

  22. RNA polymerase interacts w/promoter, regulator sequences, & enhancer sequences to begin transcription • Regulator proteins bind to regulator sequences to activate transcription • Found prior to promoter • Enhancer sequences bind activator proteins • Typically far from the gene • Silencer sequences stop transcription if they bind with repressor proteins

  23. Now, Can You: • Explain why gene expression control is necessary in a eukaryotic cell? • Describe how expression is regulated in before & during transcription? • Tell me what differentiation is? Euchromatin? A silencer sequence? • Explain how gene expression regulation is different in eukaryotes/prokaryotes?

  24. Post-Transcription Regulation • Have mRNA variation • Alternative splicing: shuffling exons • Allows various proteins to be produced in different tissues from the same gene • Change the lifespan of mRNA • Produce micro RNA that will damage mRNA, preventing translation • Edit RNA & change the polypeptide produced • Insert or alter the genetic code

  25. Translation Regulation • mRNA present in cytosol does not necessarily get translated into proteins • Control the rate of translation to regulate gene expression • How? • Modify the 5’ cap • Feedback regulation (build up of products = less translation)

  26. Translation Regulation Con’t • Modify the lifespan of proteins: • Attach ubiquitin = target for breakdown via proteasome (woodchipper)

  27. So… • What are the ways that a cell can regulate gene expression AFTER transcription? • How can the process of RNA splicing allow one pre-mRNA to produce 5 different proteins in 5 different tissues? • And…

  28. Can you accurately fill in this table?

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