1. Chapter 31 Regulation of Prokaryotic Transcription (pages 1028-1042) Learning objectives: Understand the following an operon a regulatory protein an operator negative control, positive control, catabolite repression attenuation

2. Finding and binding the promoter

3. Why is it necessary? Bacterial environment changes rapidly Survival depends on ability to adapt Bacteria must express the enzymes required to survive in that environment Enzyme synthesis is costly (energetically) Therefore, want to make enzymes when needed Transcription Regulation in Prokaryotes

4. Proteins Constitutive Always expressed “housekeeping” e.g. glucose metabolizing enzymes Glucose is the preferred carbon source for bacteriaAdaptive “inducible” Made only when needed e.g. Lactose metabolizing enzymes Made only if lactose is the sole carbon source Not made if glucose is present

5. 1. Alternate sigma factor usage: controls selective transcription of entire sets of genes     vegetative (principal  heat shock nitrogen starvation   TTGACATATAAT (16-19 bp) (5-9 bp) A +1 CNCTTGACCCATNT (13-15 bp) (5-9 bp) A +1 CTGGNA TTGCA (6 bp) (5-9 bp) A +1 Ways to Regulate Transcription

6. 2. Positive Regulation (activation): a positive regulatory factor (activator) improves the ability of RNAP to bind to and initiate transcription at a weak promoter. Ways to Regulate Transcription RNAP -35-10+1 Activator Activator binding site EXAMPLE: CAP

7. 3. Negative Regulation (repression): a negative regulatory factor (repressor) blocks the ability of RNAP to bind to and initiate transcription at a strong promoter. Ways to Regulate Transcription RNAP -35-10 Repressor Operator EXAMPLE: lac REPRESSOR

8. Protein synthesis is regulated transcriptionally Genes that encode proteins with related functions are grouped into transcriptional units called “operons” This ensures that genes for enzymes in the same metabolic pathway are all made at the same time Operons have three functional “parts” 1) structural genes: these encode proteins (usually with related functions) 2) promoter 3) regulatory sequences that interact with regulatory proteins Sometimes an operon is associated with: 4) regulatory genes: these encode proteins regulating expression of that operon

9. Structural genes promoter Operator (regulatory sequence that binds a repressor protein) Architecture of a typical operon By regulating a single promoter you can co-ordinate the expression of three genes (in this example) RNA transcript covers all genes in the operon = “polycistronic RNA”

10. Genes for enzymes for pathways are grouped in clusters on the chromosome - called operons This allows coordinated expression A regulatory sequence adjacent to such a unit determines whether it is transcribed - this is the ‘operator’ Regulatory proteins work with operators to control transcription of the genes Transcription Regulation in Prokaryotes

11. A model operon - the lac operon Structural genes Promoter (P lac ) and operator Promoter for lacI regulatory gene (lacI) In the lac operon the operator lies downstream of the promoter. In other operons a different arrangement may be found.

12. Regulation of the lac promoter - P lac AIMS: 1) turn OFF (repress) P lac in the presence of glucose 2) turn ON (induce or de-repress) P lac when lactose is the sole carbon source How is this accomplished?? VIA the lac repressor (encoded by lacI) The lac repressor is made constitutively The protein assembles into a tetramer The tetramer binds to DNA at a specific sequence found in the operator The lac repressor has high affinity for the lac operator (O lac ) BINDING TO O lac BLOCKS ELONGATION FROM P lac

13. The lac operator This is the sequence bound by the lac repressor tetramer Deduced by footprinting experiments The sequence is an inverted repeat - pallindrome (reads same forward & backward) E.g. racecar, Madam I’m Adam GGATTC CTTAGG 5’ 3’ 5’ 3’

14. The lac operator Mutations that block binding of lac repressor to operator = O c (constitutive) i.e. lac operon can’t be repressed and is therefor transcribed constitutively

15. The lac repressor protein Has a domain structure N-term = DNA binding C-term = forms multimers. C-terminal domain subunit-subunit interactions N-terminal domain DNA binding Hinge region flexible connector NN C C Limited proteolysis cleaves the protein into 2 domains at the hinge. The 2 domains can function independently

16. RNAP NN C C N N C C Transcriptional regulation RNAP elongation is blocked -5 to +21 O lac -35 -10 promoter

17. RNAP O lac lacI lacZ Transcriptional regulation Get repression with O lac alone, but get the BEST repression with additional “pseudo-operators” which bind the other half of the lac repressor tetramer and loop out the intervening DNA RNAP O lac Pseudo-operator

18. Lac repressor tetramer bound to DNA at two sites Double stranded DNA is shown in blue. One lac repressor dimer is shown in purple and green The other is shown in red and yellow The two dimers form a tetramer through interactions at the C- terminal  helices

19. Regulation of lac repressor binding to DNA This is controlled by the presence or absence of lactose in the cell (an inducer molecule) Lactose (actually an isomer called allolactose) is the natural inducer In the lab we sometimes use an artificial inducer called IPTG IPTG is not broken down in the cell so it acts as a very stable strong inducer molecule

20. Regulation of lac repressor binding to DNA Lactose IPTG

21. Lac repressor + inducer (lactose) If lactose is the sole carbon source The lac operon is induced (de-repressed) Each lac repressor monomer binds an inducer ( the binding is cooperative) conformational change Reduces the affinity of repressor for O lac RNAP can elongate and the operon is expressed! repressor dissociates from O lac

22. The lac repressor protein Has a domain structure N-term = DNA binding C-term = binds inducer, forms multimers. N N lactose C C operator lactose NN C C

23. Operon-specific control by lactose binding to lac repressor

24. The lac operon is also controlled by glucose I.e. if both lactose and glucose are present, the operon is NOT transcribed Lac repressor is not binding operator so why is operon not transcibed? Because the operon is repressed by glucose in the media Via CATABOLITE REPRESSION

25. Catabolite Repression Is a “global control system” Functions through a regulatory protein Called either: CAP (catabolite activator protein) CRP (cAMP receptor protein)

26. CAP is a positive regulator CAP binds DNA at a specific sequence CAP binds as a dimer binding is allosterically regulated by the small effector molecule, cAMP has a 2-domain structure: C-terminus binds DNA N-terminus binds cAMP and dimerizes

27. CAP Structure CAP is a 47 kDa dimer made up of two identical subunits. Each subunit has a modular, two-domain structure NN cAMP N-terminal domain subunit-subunit interactions cAMP binding positive regulation of transcription C-terminal domain DNA binding Hinge region flexible connector

28. CAP DNA binding Dimers are assembled with a 2-fold symmetry. CAP recognizes a 2-fold symmetric DNA site (“dyad-symmetric site” or “inverted repeat”) NN cAMP AA-TGTGA TT-ACACT TCACA-TT AGTGT-AA ------

29. cAMP-induced allosteric transition in CAP cAMP induces a conformational change -- an “allosteric transition”-- in CAP. This involves a change in the subunit-subunit orientation and in the domain-domain orientation Without cAMP Binds DNA with low affinity due to the proteins’ overall + charge but has no sequence-specificity NN + cAMP NN cAMP With cAMP High-affinity sequence-specific DNA binding + interaction with RNA polymerase

30. What is the connection between CAP and glucose? CAP is a glucose sensor When [glucose] is low [cAMP] increases CAP is active in DNA binding When [glucose] is high, [cAMP] decreases CAP does not bind DNA When bound to DNA CAP activates transcription Transcription of lac operon is activated when [glucose] is low

31. CAP binding sites are found near promoters of target operons CAP helps RNAP bind to promoters of target operons RNAP needs help in binding since promoter sequences are poor match to consensus CAP is a transcriptional activator

32. 5’ TTGACA spacer TATAAT 3’ Consensus 5’ TTACAC spacer TATGTT 3’ Example: P lac -35 box -10 box RNAP can’t bind to efficiently on its own CAP can contact RNAP and stabilize the formation of a closed complex on weak promoters (by 50-fold)

34. Positive Regulation (activation): a positive regulatory factor (activator) improves the ability of RNAP to bind to and initiate transcription at a weak promoter. Ways to Regulate Transcription RNAP -35-10+1 Activator Activator binding site EXAMPLE: CAP

35. CAP binding induces a bend in DNA (Contribution of the bend to transcription activation is unclear)

36. RNAP NN C C N N C C Proteins bound at lac operon regulatory region -5 to +21 -35 -10 O lac promoter -72 -51 CAP binding site CC N N cAMP

37. RNAP Glucose is sole carbon source -5 to +21 -35 -10 O lac promoter -72 -51 CAP binding site Lac operon not transcribed C N N C CAP NN C C N N C C Lac Repressor

38. RNAP Lactose is the sole carbon source O lac promoter -5 to +21 -35 -10 -72 -51 CAP binding site Lac operon is transcribed CC N N cAMP CAP N C C C C lactose N N N

39. RNAP Lactose and glucose are present -5 to +21 -35 -10 O lac promoter -72 -51 CAP binding site Lac operon is not transcribed N C C C C lactose N N N C N N C CAP

40. ConditionsCAP bound?RNAP bound?Lac rep bound? Operon transcribed? Glucose is sole carbon source XXX Glucose plus lactose XXX Lactose is sole carbon source X 4 444 Summary of lac operon regulation X

41. Induction and Repression Increased synthesis of genes in response to a metabolite is ‘induction’ e.g CAP Decreased synthesis in response to a metabolite is ‘repression’ e.g lac repressor

42. The lac Operon lac operon expresses the genes needed for lactose metabolism The structural genes of the lac operon are controlled by negative regulation lacI gene product is the lac repressor The lac operator is a palindromic DNA lac repressor - DNA binding on N-term; C-term. binds inducer, forms tetramer.

43. Catabolite Activator Protein Positive Control of the lac Operon Some promoters require an accessory protein to speed transcription Catabolite Activator Protein or CAP is one such protein CAP is a dimer of 22.5 kD peptides N-term binds cAMP; C-term binds DNA Binding of CAP-(cAMP) 2 to DNA assists formation of closed promoter complex

45. Action-at-a-distance through by protein-protein interactions between proteins bound at distant sites Mediated by DNA looping First worked out for the araBAD operon Under global control by CAP Under operon-specific control by AraC AraC binds DNA as a dimer Its allosteric effector is arabinose

46. araC is a dual-function protein araC plus arabinose is a positive regulator of transcription i.e. it contacts RNAP and helps recruit it to the weak arabinose operon promoter araC in the absence of arabinose is a negative regulator of transcription I.e. it inhibits RNAP elongation

47. Structural basis for ligand-regulated dimerization of AraC ( Science: 276 p 421 (1997) ) + arabinose promotes side- by-side dimers These bind close to RNAP and activate transcription Minus arabinose promotes head-to-head dimers

48. Effect of different AraC dimers

49. The araBAD operon

50. Regulation of the araBAD operon in the absence of arabinose

51. Regulation of the araBAD operon when arabinose is the sole carbon source

52. The trp Operon Encodes a leader sequence and 5 proteins that synthesize tryptophan Trp repressor controls the operon Trp repressor binding excludes RNA polymerase from the promoter

53. The trp Operon The trp operon is also controlled by ATTENUATION This relies on the physical coupling of transcription and translation in bacteria i.e. the ribosome starts translating a transcript BEFORE RNAP is finished synthesizing the transcript

55. The trp operon leader sequence encodes a short region of protein that is rich in trp amino acids

56. Plus Trp - want operon OFF Minus Trp - want operon ON

58. We have now finished Chapter 31 Section 3 For next class study for the TEST!!! Next Tuesday we will pick up with Chapter 31 sections 4 and 5 Pages 1043-1056 Regulation of eukaryotic transcription