Regulation of gene expression

1. Regulation of gene expression

2. Learning Outcome • To explain the regulation of gene expression in bacteria

3. Introduction • Bacteria have a remarkable ability to adapt to a rapidly changing environment • Adaptation requires that new proteins to be synthesized • Bacteria have evolved in diverse ways to coordinate gene expression with environmental signal.

4. Housekeeping genes • genes that encode such proteins as DNA polymerase, RNA polymerase, and DNA gyrase. • gene products are required under specific growth conditions • enzymes that synthesize amino acid • break down specific sugars, • or respond to a specific environmental condition such as DNA damage. • genes must be expressed at some level all of the time

5. Modes of Gene Expression • constitutive expression: gene is expressed at the same rate at all times • gene products needed no matter what the conditions might be • e.g., enzymes for glycolysis • inducible expression: gene expression changes in response to conditions • e.g., enzymes in prokaryotes for utilization of certain sugars as energy source

6. Gene Regulation • three possible places in the production of an active gene product. • as transcriptional regulation • When the gene is transcribed and how much it is transcribed influences the amount of gene product that is made. • as translational regulation • How often the mRNA is translated influences the amount of gene product that is made. • post-transcriptional or post-translational regulation mechanisms. • Both RNA and protein can be regulated by degradation to control how much active gene product is present.

7. Regulation in the cell • 2 major modes of regulation; • control the activity of preexisting enzyme • happen after protein has been synthesized • regulated by product inhibition or feedback inhibition • control the amount of enzyme synthesized • occurs at the level of transcriptionor • occurs at the level of translation

9. 1. Regulation of enzyme activity • Feedback inhibition • Involve many enzymatic steps • End product of the biosynthetic pathway inhibits the activity of the first enzyme • End product used up, synthesis can resume

11. Allosteric inhibition • Responsible for end product inhibition • Allosteric enzyme 2 binding sites • Active site- substrate binds • Allosteric site- inhibitor binds • Inhibitor binds (end product)—conformation of enzyme molecule changes--substrate no longer binds • Concentration of inhibitor fall—dissociation of inhibitor from allosteric site– active site restore shape

13. 2. Regulation of Enzyme synthesis Regulation of gene expression: • most bacterial regulation occurs at the transcriptional level. • it would be a waste to make the RNA if neither the RNA nor its encoded protein is needed. • Regulate the transcription of mRNA enzyme synthesis • Regulation of transcription • Repression • Induction

14. Regulation of gene expression DNA transcription RNA processing translation Protein

15. Prokaryotic regulation Usually no specialized cell types Can make decisions! Save energy: turn off unnecessary genes Respond quickly to changing conditions histidine in medium no histidine available make histidine Salmonella typhimurium his genes OFF his genes ON

16. How is a gene turned “on” or “off?” Decide whether or not to transcribe gene ON OFF

17. How is a gene turned “on” or “off?” Transcription: RNA polymerase (RNAP) binds promoter (-10/-35) RNAP -35 -10 gene

18. How is a gene turned “on” or “off?” • Regulatory protein • Environmental “sensor”

19. How is a gene turned “on” or “off?” 1) Regulatory protein: Binds DNA at promoter of regulated gene Repressor blocks transcription Activator encourages transcription RNAP RNAP RNAP RNAP RNAP RNAP RNAP RNAP RNAP RNAP -35 -10 gene

20. How is a gene turned “on” or “off?” 1) Regulatory protein: Binds DNA at promoter of regulated gene Repressor blocks transcription Activator encourages transcription RNAP repressor binding site (operator) -35 -10 gene

21. How is a gene turned “on” or “off?” 1) Regulatory protein: Binds DNA at promoter of regulated gene Repressor blocks transcription Activator encourages transcription RNAP RNAP RNAP RNAP RNAP RNAP RNAP RNAP RNAP RNAP activator binding site -35 -10 gene

22. How is a gene turned “on” or “off?” 2) Environmental “sensor” Small molecule Activates or inactivates regulatory protein sensor sensor inactive repressor active activator active repressor inactive activator -35 -10 gene

23. Enzyme Repression • repression- inhibit gene expression and decreases the synthesis of enzymes • response to abundance of end-product • e.g. arginine synthesized only when arginine is not present in culture medium

24. mediated by repressors ( block the ability of RNA polymerase to initiate transcription) • repressor requires a corepressor in order to bind to operator site • corepressor controls enzyme synthesis (involved in the biosynthetic pathways)

26. Enzyme Induction • Induction-turns on the transcription of genes - synthesis of the enzyme only when its substrate is present • inducer- a substance that initiate enzyme induction • inducible enzyme- enzymes synthesized in the presence of inducer • e.g. production of b-galactosidase by E. coli in the presence of lactose, so lactose can be metabolized

28. Operon model of gene expression • Genes which determined the structures of proteins (Enzymes) : structural genes • Operon: a group of coordinately regulated structural genes with related metabolic functions and thepromoterandoperatorsites that control their transcription • promoter; the region of DNA where RNA polymerase initiates transcription • operator; the region of DNA adjacent to structural genes that controls their transcription • regulatory gene codes for repressor protein

30. Operons Related genes often transcribed together One promoter, one terminator Shine-Dalgarno sequences allow multiple genes on one RNA promoter terminator trpE trpE trpD trpD trpC trpC trpB trpB trpA trpA enzymes needed to synthesize tryptophan genes needed to synthesize tryptophan transcription translation mRNA Shine-Dalgarno sequences DNA

31. Mechanism of repression • inducer (lactose) absence • Corepressor (eg. Arginine) combine with repressor (structure altered by this combination) • repressor binds to operator • no mRNA synthesized • no enzymes

33. trp Operon If there is no tryptophan available, should the trp operon be on (transcribed) or off?

34. trp Operon Transcribed while tryptophan level is low ON

35. trp Operon If tryptophan is present: Tryptophan (sensor) binds repressor protein Repressor becomes active and binds operator site OFF

36. trp Operon Feedback inhibition of metabolic pathway “Feedback inhibition” of transcription tryptophan 1 2 3 4 5 Enzyme A Enzyme B Enzyme C Enzyme D Enzyme E allosteric inhibitor repressor RNAP trpE trpD trpC trpB trpA operator allosteric activator

37. Mechanism of induction • inducer presence (lactose-- allolactose) • it binds to repressor, inactivates it • operator unbound • mRNA synthesized • enzyme synthesis induced

39. lac Operon Glucose is E. coli’s preferred energy source Bacteria grow faster in glucose (readily catabolizable) than lactose (when either one as a sole carbon source). Can use lactose, if available Requires b-galactosidase and two other enzymes

40. Growth rate of E. coli on glucose and lactose alone glucose No of cells (log) lactose Time

41. Growth rate of E. coli on glucose and lactose combined All glucose consumed glucose used lactose used No of cells (log) lag time Time

42. Cast of characters: lac Operon operator allolactose CAP (inactive activator) repressor (active) cyclic AMP (cAMP) cap lacI genes for using lactose C O lacZ lacY lacA P P P CAP site promoter regulatory proteins environmental sensors

43. If no lactose is available: lac Operon CAP (inactive activator) cap lacI RNAP C O lacZ lacY lacA P P P repressor (active) OFF

44. If lactose becomes available: lac Operon CAP (inactive activator) cap lacI repressor (inactive) allolactose RNAP RNAP C O lacZ lacY lacA P P P OFF (ON)

45. Regulation of lactose operon depend on glucose in the medium • Which in turn control cyclic AMP (cAMP) • cAMP- serves as cellular alarm signal

46. glucose available, cell used glucose first • glucose not available- cell produced cAMP • cAMP bind to CAP (catabolite activator) • Complex (cAMP + CAP) binds to lac promoter-help in binding of RNA polymerase to the promoter • transcription of structural genes--cell now grow on lactose

47. If lactose is available and glucose is low: lac Operon cap lacI repressor (inactive) CAP (active) RNAP C O lacZ lacY lacA P P P cAMP CAP (inactive activator) ON (ON)

48. Overview and General Features • Organisms may not wish to express all genes at all times • inefficient and wasteful of energy • prokaryotes: • expression of genes as needed • dictated by environment • eukaryotes: • expression of genes as needed • expression of genes at particular times and in particular types of cells • regulation of gene expression during development