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Molecular Biology and Biochemistry 694:408 / 115:512 Spring 2007, Lectures 13-14 Regulation of prokaryotic transcripti

Molecular Biology and Biochemistry 694:408 / 115:512 Spring 2007, Lectures 13-14 Regulation of prokaryotic transcription Watson et al., (2004) Mol. Biol. Of the Gene, Chapter 16 Garrett and Grisham, Biochemistry (2005), Chapter 29 (pg. 942-974)

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Molecular Biology and Biochemistry 694:408 / 115:512 Spring 2007, Lectures 13-14 Regulation of prokaryotic transcripti

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  1. Molecular Biology and Biochemistry 694:408 / 115:512 Spring 2007, Lectures 13-14 Regulation of prokaryotic transcription Watson et al., (2004) Mol. Biol. Of the Gene, Chapter 16 Garrett and Grisham, Biochemistry (2005), Chapter 29 (pg. 942-974) Lodish et al., (2000) Mol. Cell Biol. Chapter 10 (pg. 342); Chapter 12 (pg. 485-491) Lewin (2000), Genes VII, Chapter 9; Chapter 10

  2. Strong promoters contain close matches to the consensus site

  3. Transcription from some promoters is initiated by alternative sigma () factors

  4. Different  factors in Bacillus subtilis are used at different stages of growth (vegetative vs. sporulation) Sigma Source & Use -35 region -10 region s43 vegetative: general genes TTGACA TATAAT s28 vegetative: flagellar genes CTAAA CCGATAT s37 used in sporulation AGGNTTT GGNATTGNT s32 used in sporulation AAATC TANTGTTNTA s29 synthesized in sporulation TTNAAA CATATT gp28 SPO1 middle expression AGGAGA TTTNTTT gp33-34 SPO1 late expression CGTTAGA GATATT

  5. Different  factors in Bacillus subtilis are used at different stages of growth (vegetative vs. sporulation) Sigma Source & Use -35 region -10 region s43 vegetative: general genes TTGACA TATAAT s28 vegetative: flagellar genes CTAAA CCGATAT s37 used in sporulation AGGNTTT GGNATTGNT s32 used in sporulation AAATC TANTGTTNTA s29 synthesized in sporulation TTNAAA CATATT gp28 SPO1 middle expression AGGAGA TTTNTTT gp33-34 SPO1 late expression CGTTAGA GATATT

  6. Bacteriophage - "eaters of bacteria"

  7. Transcription of phage SPO1 genes 70 70 28 RNAP RNAP RNAP RNAP RNAP RNAP RNAP 28 28 28 34 33 34 34 33 33 Phage Early gene 28 Early Phage Mid. genes 33 34 Middle Phage Late genes Late

  8. Genetic regulation lac system of E. coli “What’s true for E. coli is true for an elephant.” J. Monod

  9. b-Gal is produced only when lactose is present

  10. b-gal induction can be due to 1. Activation of preexisting enzyme (i.e., removal of repressor) 2. Synthesis of new enzyme

  11. Lactose is both an inducer and a substrate for b-Gal Gratuitous inducers do not act as substrates Some substrates do not work as inducers Action of the enzyme on the inducer is neither necessary nor sufficient for induction

  12. Induction kinetics of b-Gal under gratuitous conditions p = (amount of b-Gal)/(total cell protein)

  13. lac system: transcription regulation

  14. RNAP 1 mRNA 2 Regulation of Transcription 1. Transcription initiation/RNA synthesis 2. mRNA Turnover

  15. Selection of Lac- mutants (negative selection nutritional marker) +Lac

  16. 1 2 Tricks use chromogenic substrates (X-gal) and gratuitous inducers (IPTG) to select for Lac mutants (Lac+ - blue, Lac- - white) use diagnostic plates (EMB) to elect for absence of sugar fermentation

  17. The lac locus of E. coli galactoside permease b-Gal galactoside transacetylase lacZ mutants are Lac- lacY mutants are cryptic lacA mutants are Lac+ lacI mutants are constitutive (first example of mutants that affect production, not activity)

  18. The PaJaMo experiment Set a cross in the absence of inducer: Hfr lacI+lacZ+ StrS TsXS x F- lacI-lacZ- StrR TsxR After some time, kill the donor with Str and T6 Monitor b-Gal in the presence or in the absence of inducer

  19. The properties of lacO mutants provide genetic proof of operon model

  20. lac operator Most bacterial operator sequences are short inverted repeats; Most transcription regulators are dimeric

  21. The presence of inducer changes the conformation of LacI repressor so that it can no longer bind DNA

  22. Distinction between factors (proteins) and elements (DNA sites) i) Regulatory factors act in trans ii) Regulatory elements act in cis

  23. The LAC OPERON

  24. LacI binds DNA as a tetramer to better repress transcription Why did Jacob & Monod not find O2 and O3?

  25. Genetic analysis of the LacI binding sites X-gal White 4 4 0 Blue O O O 1 3 2 White 7 0 0 O O O 1 3 2 White 1 8 O O O 1 3 2 1 . 9 O O O 1 3 2 Blue R e p r e s s i o n P l a c Z 1 3 0 0 O O O 1 3 2 1 . 0 O O O 1 3 2 1 . 0 O O O 1 3 2 1 . 0 O O O 1 3 2

  26. Glucose effect: no response to inducers in the presence of glucose

  27. Catabolism ??? glucose energy pgi glycerol pgi- mutants grown on glycerol induce lac genes even in the presence of glucose Interpretation: glucose effect is due a product of glucose catabolism (catabolic repression)

  28. Catabolite repression occurs for a wide range of sugars Catabolite repression mutants must therefore be defective in utilization of wide range of sugars (cells will be permanently repressed). Select on EMB agar.

  29. Mutants defective in catabolite regulation occur in two distinct loci cya crp codes for CAP (catabolite activating protein). CAP, when bound to cAMP, binds to lac regulatory region and activates transcription of structural genes cAMP level high when glucose is low

  30. LAC Operon and catabolite repression Positive control of the lac operon is exerted by cAMP-CAP Catabolite Activator Protein

  31. Cooperative binding of cAMP-CAP and RNA polymerase to the lac control region activates transcription

  32. The lac control region contains three critical cis-acting sites RNAP CAP RNAP LacI

  33. lac operator: the regulatory region

  34. Residues that interact with RNAP CAP binding bends the DNA

  35. Operator sites can be in different places with respect to the start of the promoter

  36. Different mechanisms of transcriptional activation A) Strong promoters B) Promoters with UP elements C) Activation through interactions with the aCTD D) Activation through interactions with other components of RNAP E) Activation through interactions with components multiple components of RNAP by multiple activators

  37. Different types of negative and positive control of transcription

  38. Changes in DNA topology affect isomerization step in formation of the open complex

  39. Mechanism of activation by MerR RNAP RNAP merT MerR -10 -35 19 bp Hg++ MerR merT -10 -35 17 bp Average Prom. -10 -35 15-17 bp

  40. Enzyme repression: the trp operator The synthesis of Trp structural genes is controlled by unlinked TrpR repressor. TrpR binds to Trp operator in the presence of Trp (product inhibition). Both trpR and trpO mutants are derepressed

  41. Crossfeeding analysis of Trp mutants allows to analyze the biochemistry of Trp biosynthesis pathway TrpE TrpD TrpB precursor Trp

  42. Attenuation of trp operator expression attenuator Deletions in the attenuator increase basal synthesis of Trp enzymes

  43. the trp attenuator region

  44. Attenuation occurs due to formation of alternative secondary RNA structures in the leader sequence in the presence or absence or Trp

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