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Chapter 31

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Chapter 31

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  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. Transcription Regulation in Prokaryotes • 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

  4. Proteins Constitutive • Always expressed • “housekeeping” • e.g. glucose metabolizing enzymes • Glucose is the preferred carbon source for bacteria Adaptive • “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. s70 Ways to Regulate Transcription 1. Alternate sigma factor usage: controls selective transcription of entire sets of genes vegetative (principal s) +1 (16-19 bp) (5-9 bp) A TTGACA TATAAT heat shock +1 s32 (13-15 bp) (5-9 bp) A CNCTTGA CCCATNT +1 nitrogen starvation s60 (6 bp) (5-9 bp) TTGCA A CTGGNA

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

  7. Ways to Regulate Transcription 3. Negative Regulation (repression): a negative regulatory factor (repressor) blocks the ability of RNAP to bind to and initiate transcription at a strong promoter. RNAP Repressor -35 -10 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. promoter Structural genes RNA transcript covers all genes in the operon = “polycistronic RNA” 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)

  10. Transcription Regulation in Prokaryotes • 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

  11. Promoter (Plac) and operator Structural genes Promoter for lacI regulatory gene (lacI) A model operon - the lac operon 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 - Plac AIMS: 1) turn OFF (repress) Plac in the presence of glucose 2) turn ON (induce or de-repress) Plac 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 (Olac) BINDING TO Olac BLOCKS ELONGATION FROM Plac

  13. The lac operator E.g. racecar, Madam I’m Adam 5’ 5’ 3’ 3’ GGATTC CTTAGG • 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)

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

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

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

  17. Transcriptional regulation Olac RNAP RNAP lacI lacZ Pseudo-operator Pseudo-operator Olac Get repression with Olacalone, 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

  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 a 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 IPTG Lactose

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

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

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

  28. AA-TGTGA TT-ACACT TCACA-TT AGTGT-AA ------ ------ 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”) N N cAMP cAMP

  29. N N N N cAMP cAMP + cAMP With cAMP High-affinity sequence-specific DNA binding + interaction with RNA polymerase Without cAMP Binds DNA with low affinity due to the proteins’ overall + charge but has no sequence-specificity 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

  30. 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 What is the connection between CAP and glucose?

  31. CAP is a transcriptional activator • 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

  32. Example: Plac 5’ TTACAC spacer TATGTT 3’ -10 box -35 box 5’ TTGACA spacer TATAAT 3’ Consensus • 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)

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

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

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

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

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

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

  39. Summary of lac operon regulation Operon transcribed? Conditions CAP bound? RNAP bound? Lac rep bound? Glucose is sole carbon source X X 4 X Glucose plus lactose X X X X Lactose is sole carbon source 4 4 4 X

  40. 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

  41. 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.

  42. Catabolite Activator ProteinPositive 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

  43. 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

  44. 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

  45. 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

  46. Effect of different AraC dimers

  47. The araBAD operon

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