REGULATION OF GENE EXPRESSION PROKARYOTES

1. REGULATION OF GENE EXPRESSIONPROKARYOTES

2. 3 LEVELS OF GENE EXPRESSION REGULATION

3. OVERVIEW OF REGULATORY MECHANISMS

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

6. REGULATION OF GENE EXPRESSION • REGULATION OF ENZYMATIC ACTIVITY

7. ENZYME REGULATION DURING METABOLISM CONTROL OF ENZYMATIC ACTIVITY

8. REGULATION BY FEEDBACK INHIBITION

9. FEEDBACK INHIBITION • ISOLEUCINE SYNTHETIC PATHWAY

10. CATABOLIC OPERONS INDUCIBLE ENZYMES

11. 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". Journal of Bacteriology. 1 March 2004. Retrieved 30 December 2012.

12. CONTROL OF GENE EXPRESSION • OPERON MODEL-François Jacob and JaquesMonod (1961)-Regulated genes can be switched on/off depending on cell's metabolic needs • Basic Definitions • Operon = A regulated cluster of adjacent structural genes with related functions • 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 . Prokaryotic transcription unit - 5 or more genes • Transcription —> Long mRNA molecule • Translation —> separate polypeptides

13. lac OPERON

14. CONTROL OF GENE EXPRESSION OPERONoperons.swf • 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

15. CONTROL OF GENE EXPRESSION IN PROKARYOTESTHE 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

16. CONTROL OF GENE EXPRESSION IN PROKARYOTESTHE LACTOSE UTILIZATION OPERON • Structural Genes • lacZ-b-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

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

18. LAC REPRESSOR INTERACTING WITH DNA

19. (Constitutive synthesis)

20. OVERVIEW • POSITIVE & NEGATIVE CONTROL OF GENE EXPRESSION

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

23. lac OPERON • 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

25. lac OPERON • 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

26. 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

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

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

30. 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. colieconomizes 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)

31. 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

32. ANABOLIC OPERONS REPRESSIBLE ENZYMES

33. 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

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

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

37. 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