29 105 fa13 control of gene expression prokaryotes skel


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  • 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
  • 29 105 fa13 control of gene expression prokaryotes skel

    1. 1. Control of Gene Expression: Prokaryotes BIOL 105 Dr. Corl October 30, 2013
    2. 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. 3. Regulation of Gene Expression • Gene expression can be regulated: – During transcription (transcriptional control). – During translation (translational control). – After translation (post-translational control).
    4. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 19. Summary Thus Far
    20. 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. 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. 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. 23. The lac and trp Operons
    24. 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. 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. 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. 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. 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. 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. 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. 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. 32. Glucose Regulates cAMP Levels • In this way, glucose, by regulating cAMP levels, can regulate the expression of the lac operon genes.
    33. 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. 34. Summary: The lac Operon
    35. 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?