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29 105 fa13 control of gene expression prokaryotes skel

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  • Gene= lacZ---PROTEIN=B-GALACTOSIDASE FUNCTION=BREAK DOWN LACTOSE
    Gene= lacY---GAL PERMEASE. FUNTION= IMPORT LACTOSE INTO CELL
  • lacI- lacI repressor protein---binds to DNA and inhibits transcription of lacZ and lacY when lactose levels are low(its normal job)
  • lacI binds in a way that it inhibits transcription
  • Transcript

    • 1. Control of Gene Expression: Prokaryotes BIOL 105 Dr. Corl October 30, 2013
    • 2. Gene Expression • We can say that Gene “A” is being expressed if: – Gene “A” is being transcribed to form mRNA – That mRNA is being translated to form proteins – Those proteins are properly folded and are in state where they can be used by the cell • Cells are extremely selective about what genes are expressed, in what amounts, and when. • Gene expression can be regulated at a variety of different stages.
    • 3. Regulation of Gene Expression • Gene expression can be regulated: – During transcription (transcriptional control). – During translation (translational control). – After translation (post-translational control).
    • 4. Some Examples of Regulation of Gene Expression • Transcriptional control: – Regulatory proteins affect the ability for RNA polymerase to bind to or transcribe a particular gene. • Translational control: – Various proteins may affect rate of translation. – Enzymes may affect lifetime of an mRNA transcript. • Post-translational control: – Translated protein may be modified by phosphorylation, which may change its folding and/or activity.
    • 5. Gene Expression • Some genes are constitutively expressed: – Expressed equally at all times. • Many other genes are regulated and their expression may be induced or repressed: – Gene expression is not just “on” or “off” it can vary along a continuum! • Today we will be looking at transcriptional regulation of gene expression in bacteria.
    • 6. Lactose Metabolism in E. coli • Bacteria break down lactose into its component monomers, glucose and galactose, using the enzyme β-galactosidase. • Bacteria can then use the monomers to generate ATP through cellular respiration.
    • 7. Lactose Metabolism in E. coli • Lactose gets transported (imported) into the cell with the help of galactoside permease. • Once inside the cell, lactose can be cleaved into monomers with the help of β-galactosidase.
    • 8. Lactose Metabolism in E. coli • The expression of the gene for β-galactosidase (and galactoside permease) is regulated by lactose and glucose: – High levels of LACTOSE stimulate (induce) the expression of the gene for β-galactosidase. – High levels of glucose inhibit (repress) the expression of the gene for β-galactosidase. • How (by what mechanisms) do lactose and glucose regulate gene expression in E. coli?
    • 9. Lactose Metabolism Mutants • Monod and Jacob conducted a genetic screen for E. coli mutants that were specifically defective in lactose metabolism: – Generated a large number of E. coli colonies bearing mutations in random locations in their genomes. – “screened” through these colonies to find ones that could not successfully grow on a “lactose only” medium, but could grow on a “glucose only” medium.
    • 10. Screen Results • Three classes of E. coli mutants defective in lactose metabolism were found: – 1.) lacZ- mutants lacked functional β-galactosidase. – 2.) lacY- mutants lacked the membrane protein galactosidase permease.
    • 11. Screen Results • 3.) lac I- mutants: – Produce β-galactosidase and galactoside permease even when lactose is absent. – lacI- mutants are constitutive mutants: • They produce product(s) at all times. • Loss of regulation by lactose. – The lacI gene must code for a protein that normally serves to repress expression of the lacZ and lacY genes when lactose is absent.
    • 12. Screen Conclusions • The lacZ gene codes for: β-galactosidase. • The lacY gene codes for: – Galactoside permease. • The lacI gene codes for: – A regulatory protein (a repressor) that normally functions to repress lacZ and lacY gene expression when lactose is absent.
    • 13. The lac Genes • The lacZ, lacY, and lacI genes are in close physical proximity to each other on the bacterial chromosome. • How does the lacI gene product repress gene expression of lacZ and lacY?
    • 14. Negative Control of Transcription • Negative control occurs when: – A regulatory protein (a repressor) binds to DNA and decreases the rate of transcription of downstream genes. • The lacI gene codes for a repressor, a regulatory protein that exerts negative control over the lacZ and lacY genes.
    • 15. Positive Control of Transcription • Positive control occurs when: – A regulatory protein (an _______) binds to DNA and ________ the rate of transcription of downstream genes. • We haven’t encountered this yet, but we will by the end of this lecture. Stay tuned!
    • 16. Negative Control of lacZ and lacY Gene Expression • The lacI gene codes for a repressor that binds to DNA just downstream of the promoter, physically blocking transcription of lacZ and lacY.
    • 17. Negative Control of lacZ and lacY Gene Expression • In the presence of lactose: – – – – Lactose binds to the repressor. Lactose-repressor complex releases from DNA. RNA polymerase can now transcribe lacZ and lacY. Thus, lactose induces transcription by preventing the repressor from exerting negative control.
    • 18. Negative Control of lacZ and lacY Gene Expression • If the lacI gene is mutated (lacI- mutants): – No functional repressor is synthesized. – No repressor is ever present in the cell to bind downstream of the promoter to block transcription. – lacZ and lacY will be transcribed regardless of whether lactose is present or absent.
    • 19. Summary Thus Far
    • 20. The lac Operon • Operon: – A set of coordinately regulated bacterial genes. • The lac operon: – A group of genes involved in lactose metabolism. – Also includes the lacA gene, which codes for a protective enzyme, transacetylase. – Encodes a polycistronic mRNA: • mRNA that contains >1 protein encoding segment. – The lacI protein is a repressor that binds to the operator region of the lac operon. – Lactose is an inducer, binding to the lacI repressor protein and causing it to release from the operator.
    • 21. The trp Operon • Contains 5 genes coding for proteins (enzymes) required for the synthesis of the amino acid tryptophan. Also contains a promoter and operator. • When tryptophan levels in the cell are low, the expression of the trp operon genes is relatively high. – There are trp repressor proteins in the cell, but they are unable to bind to the trp operon operator all on their own.
    • 22. The trp Operon • When tryptophan levels in the cell are high: – Tryptophan binds to the repressor and activities it. – The activated trp repressor can now bind to the trp operator and block transcription of the trp operon genes.
    • 23. The lac and trp Operons
    • 24. Positive Control of Transcription • Occurs when a regulatory protein (an activator) binds to DNA and increases the rate of transcription of downstream genes. • Let’s look at two examples of activator proteins at work: – AraC proteins can increase transcription of the ara operon genes. – CAP proteins can increase transcription of the lac operon genes.
    • 25. Positive Control of the ara Operon • E. coli can also utilize arabinose, a pentose found in plant cell walls, as an energy source. • The ara operon includes: – 3 genes required for arabinose metabolism. – A promoter to which RNA polymerase can bind. – An initiator sequence to which an activator protein (AraC) can bind to stimulate transcription.
    • 26. Positive Control of the ara Operon • Expression of the ara operon genes is high only when arabinose levels are high: – (If you were E. coli, you wouldn’t want to waste precious resources expressing these genes unless there was arabinose around to break down!) – How does the presence of arabinose stimulate expression of the ara operon genes?
    • 27. Positive Control of the ara Operon • If arabinose levels are relatively high… – Arabinose binds to AraC proteins in the cell… – ….which allows the AraC proteins to bind to the initiator region of the ara operon…. – ….which helps RNA polymerase to bind to the promoter region of the ara operon successfully… – …which increases transcription rate of ara operon genes.
    • 28. Positive Control of the ara Operon • Thus, AraC proteins, when bound to arabinose, can act as activators of gene expression at the ara operon: – Example of positive control of gene expression. • Let’s look at one more example of positive control of gene expression, this time going back to the lac operon. – Gene expression at the lac operon is under both: • Negative control (by the lacI repressor protein) • Positive control (by a protein called CAP)
    • 29. Positive Control of the lac Operon • Binding of CAP (an activator protein) to the CAP site (a DNAregion) just upstream of the lac operon promoter greatly increases lac operon gene expression. • CAP can only bind the CAP site if CAP is bound to cAMP (cyclic AMP).
    • 30. Positive Control of the lac Operon • If cAMP levels are low, CAP is inactive and won’t bind to the CAP site, and therefore transcription will be infrequent. • What regulates cAMP levels?
    • 31. Glucose Regulates cAMP Levels • cAMP is synthesized from ATP by an enzyme called adenylyl cyclase. • Glucose inhibits adenylyl cyclase activity. • If glucose levels are high, this will cause cAMP levels in the cell to be low.
    • 32. Glucose Regulates cAMP Levels • In this way, glucose, by regulating cAMP levels, can regulate the expression of the lac operon genes.
    • 33. Another Effect of Glucose • Recent studies have revealed a second, perhaps even more significant, contribution of glucose to regulating lac operon expression: – High level of glucose leads to a dephosphorylation and inactivation of gal. permease in E. coli cell membrane… – …which inhibits lactose transport into the cell… – ….which prevents lactose accumulation inside the cell… – …which allows the lacI repressor protein to bind to the lac operator, decreasing lac operon gene expression.
    • 34. Summary: The lac Operon
    • 35. Review Questions • What is meant by “gene expression?” At what levels can gene expression be controlled? • Draw out the lac operon. How is it negatively controlled? Positively controlled? • Contrast the regulation of the trp operon versus the regulation of the lac operon. • How is the ara operon regulated by arabinose and the AraC protein?