2. REGULATION OF GENE EXPRESSION PROKARYOTES

3. 3 LEVELS OF GENE EXPRESSION REGULATION

4. OVERVIEW OF REGULATORY MECHANISMS

5. CONTROL OF GENE EXPRESSION CONTROL OF GENE EXPRESSION IN PROKARYOTES  Enables bacteria to adjust their metabolism to environmental change  Responses to environmental stimuli

7. REGULATION OF GENE EXPRESSION REGULATION OF ENZYMATIC ACTIVITY

8. ENZYME REGULATION DURING METABOLISM CONTROL OF ENZYMATIC ACTIVITY

9. REGULATION BY FEEDBACK INHIBITION

10. FEEDBACK INHIBITION ISOLEUCINE SYNTHETIC PATHWAY

11. CATABOLIC OPERONS INDUCIBLE ENZYMES

12. CONTROL OF GENE EXPRESSION CONTROL OF GENE EXPRESSION IN PROKARYOTES »Enzyme synthesis (Regulation of gene expression) At the level of transcription of the genes coding for particular enzymes - control the # of enzyme molecules produced Slower to take effect than feedback inhibition, but is more economical for the cell. It prevents unneeded protein synthesis for enzymes, as well as, unneeded pathway product »Examples illustrating regulation of a metabolic pathway is the tryptophan pathway in E. coli. Mechanisms for gene regulation were first discovered for E. coli »Current understanding of such regulatory mechanisms at the molecular level is primarily limited to bacterial systems »Reports on some eukaryotes & viruses* *Displacements of Prohead Protease Genes in the Late Operons of Double-Stranded-DNA Bacteriophages"*Displacements of Prohead Protease Genes in the Late Operons of Double-Stranded-DNA Bacteriophages". Journal of Bacteriology. 1 March 2004. Retrieved 30 December 2012.

13. CONTROL OF GENE EXPRESSION OPERON MODEL-François Jacob and Jaques Monod (1961)-Regulated genes can be switched on/off depending on cell's metabolic needs  Basic Definitions »Operon = A group of clustered genes that produces a single messenger RNA molecule in transcription and that consists of structural genes and regulating elements »Structural gene = Gene that codes for a polypeptide Common in bacteria and phages Has a single promoter region, so RNA polymerase will transcribe all structural genes on an all/none basis Transcription produces a single polycistronic mRNA with coding sequences for all enzymes in a metabolic pathway Translation —> separate polypeptides

14. lac OPERON

15. CONTROL OF GENE EXPRESSION OPERON MODEL-François Jacob and Jaques Monod (1961)  Basic Definitions »Polycistronic mRNA = A large mRNA molecule that is a transcript of several genes Is translated into separate polypeptides Contains stop and start codons for the translation of each polypeptide »Grouping structural genes into operons provides an advantage b/c: Expression of all genes can be coordinated. When a cell needs the product of a metabolic pathway, all the necessary enzymes are synthesized at one time. The entire operon can be controlled by a single operator OPERONoperons.swfoperons.swf

16. CONTROL OF GENE EXPRESSION IN PROKARYOTES THE LACTOSE UTILIZATION OPERON  Basic Definitions »Inducible operon »Operator-(between promoter and structural genes/within promoter)-controls access to RNA polymerase to the structural genes »Repressor protein-binds to the operator and blocks the attachment of RNA polymerase to the promoter Repressor protein encoded by regulatory gene Corepressor-usually observed in biosynthetic operons (trp operon) »Structural genes »lac operon-a catabolic operon lacI-regulatory gene-encodes repressor protein

17. CONTROL OF GENE EXPRESSION IN PROKARYOTESTHE LACTOSE UTILIZATION OPERON  Structural Genes »lacZ-  -galactosidase ( lactose Glucose + Galactose) »lacY-permease »lacA-transacetylase  Inducer-allolactose (an isomer of lactose) »Inducer present-operon active-synthesis of enzymes for metabolism of lactose »Inducer absent-operon inactive-active repressor binding to the operator prevents access to RNA polymerase Basal levels of lactose metabolic enzymes due to unstable interaction between repressor protein and operator  AN EXAMPLE OF NEGATIVE REGULATION

18. (b) Lactose present, repressor inactive, operon on (a) Lactose absent, repressor active, operon off mRNA Protein DNA mRNA 5 Protein Active repressor RNA polymerase Regulatory gene Promoter Operator mRNA 5 3 Inactive repressor Allolactose (inducer) 5 3 No RNA made RNA polymerase Permease Transacetylase lac operon  -Galactosidase lacY lacZ lacAlac I lacZ Animation Ch. 8 Operons Induction lac OPERON

19. LAC REPRESSOR INTERACTING WITH DNA

20. (Constitutive synthesis)

21. OVERVIEW POSITIVE & NEGATIVE CONTROL OF GENE EXPRESSION

22. This one Lac operon: CAP (Catabolic activator protein)

24. POSITIVE REGULATION OF THE LAC OPERON  Positive control of a regulatory system occurs only if an activator molecule interacts directly with the genome to turn on transcription (lac operon)  lac operon is under dual regulation that includes negative control by repressor protein and positive control by cAMP receptor protein (CAP)  CAP (gen: crp) = An allosteric protein that binds cAMP and activates transcription binding to an operon's promoter region (enhances the promoter's affinity for RNA polymerase)  cAMP-CAP-positive activator of lactose metabolic enzyme synthesis (facilitates RNA pol. binding to the promoter-if glucose is absent  Glu absent: cAMP high Glu present: cAMP low lac OPERON

26. POSITIVE CONTROL  E. coli preferentially uses glucose over lactose as a substrate for glycolysis (Higher efficiency)  Therefore, normal expression of the lac operon requires: »Presence of lactose »Absence of glucose (crp:cAMP receptor protein gene )  When Glu concentration decreases, cAMP increases

27. lac OPERON POSITIVE CONTROL How is CAP affected by the absence or presence of glucose?  When glucose missing, cell accumulates cAMP, a nucleotide derived from ATP.  cAMP activates CAP so that it can bind to the lac promoter  When glucose concentration rises, glucose catabolism decreases the cAMP concentration

28. POSITIVE REGULATION OF THE lac OPERON [GLUCOSE] [cAMP] rises cAMP binds CAP cAMP-CAP complex binds lac promoter Efficient transcription of lac operon [cAMP] becomes scarce cAMP loses CAP CRP disengages from lac promoter Slow transcription of lac operon Absent Present

30. lac OPERON lac operonhttp://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.htmlhttp://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html * lac operonhttp://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter12/animation_quiz_4.htmlhttp://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter12/animation_quiz_4.html lac operonoperons_induction.swfoperons_induction.swf Self quiz link: http://highered.mcgraw- hill.com/sites/0072556781/student_view0/chapter12/animation_quiz_4.htm l

31. DUAL REGULATION OF lac OPERON IN THIS TYPE OF REGULATION  Negative control by repressor determines whether or not the operon will transcribe the structural genes  Positive control by CRP determines the rate of transcription (slow vs. efficient )  E. coli economizes on RNA/protein synthesis with the help of these negative and positive controls  CRP is an activator of several different operons that program catabolic pathways  Glucose's presence deactivates CRP. This, slows the synthesis of those enzymes a cell needs to use catabolites other than glucose  E. coli preferentially uses glucose as its primary carbon and energy source, and the enzymes for glucose catabolism are coded for by unregulated genes that are continuously transcribed (constitutive)

32. DUAL REGULATION OF THE lac OPERON IN THIS TYPE OF REGULATION  Therefore, when glucose is present, CRP does not work and the cell's systems for using secondary energy sources are inactive  When glucose is absent, the cell metabolizes alternate energy sources »The cAMP level rises, CRP is activated and transcription begins of operons that program the use of alternate energy sources (e.g., lactose) »Which operon is actually transcribed depends upon nutrient availability Example: If lactose is present, the lac operon will be switched on as allolactose inactivates the repressor

33. ANABOLIC OPERONS REPRESSIBLE ENZYMES

34. REPRESSIBLE OPERONS REPRESSIBLE ENZYMES  Their synthesis is inhibited by the specific metabolite »trp operon trp present-operon inactive-trp is the corepressor trp absent-operon active BACTERIA ARE REMARKABLE IN THEIR ABILITY TO ADAPT TO A VARIETY OF ENVIRONMENTSS BY THEIR ELABORATE CONTINGENCY OF MECHANISMS TO CONTROL ENZYME SYNTHESIS AND HENCE METABOLIC PATHWAYS

35. Polypeptide subunits that make up enzymes for tryptophan synthesis (b) Tryptophan present, repressor active, operon off Tryptophan (corepressor) (a) Tryptophan absent, repressor inactive, operon on No RNA made Active repressor mRNA Protein DNA mRNA 5 Protein Inactive repressor RNA polymerase Regulatory gene Promoter trp operon Genes of operon Operator Stop codon Start codon mRNA trpA 5 3 trpR trpE trpD trpC trpB ABC D E

37. trp OPERON Animation Ch. 8 Operons repression trp operon http:// highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html http:// highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html Additional regulation mechanism: Attenuation of trp operon: http://www.youtube.com/watch?v=8aAYtMa3GFU

38. REPRESSIBLE VS. INDUCIBLE OPERONS REPRESSIBLE (trp)  Their genes are switched on until metabolite activates the repressor  They function in anabolic pathways  Pathway’s end product switches off its own production by repressing enzyme synthesis ANABOLIC INDUCIBLE (Lac)  Their genes are switched off until a specific metabolite inactivates the repressor  They function in catabolic pathways  Enzyme synthesis is switched on by the nutrient the pathway uses CATABOLIC