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 s 43 vegetative: general genes TTGACATATAAT s 28 vegetative: flagellar genesCTAAACCGATAT s 37 used in sporulationAGGNTTTGGNATTGNT s 32 used in sporulationAAATCTANTGTTNTA s 29 synthesized in sporulationTTNAAACATATT gp 28 SPO1 middle expressionAGGAGATTTNTTT gp 33-34 SPO1 late expressionCGTTAGAGATATT

5. Different  factors in Bacillus subtilis are used at different stages of growth (vegetative vs. sporulation) Sigma Source & Use-35 region-10 region s 43 vegetative: general genes TTGACATATAAT s 28 vegetative: flagellar genesCTAAACCGATAT s 37 used in sporulationAGGNTTTGGNATTGNT s 32 used in sporulationAAATCTANTGTTNTA s 29 synthesized in sporulationTTNAAACATATT gp 28 SPO1 middle expressionAGGAGATTTNTTT gp 33-34 SPO1 late expressionCGTTAGAGATATT

6. Bacteriophage - "eaters of bacteria"

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

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

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

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

11. Gratuitous inducers do not act as substrates Lactose is both an inducer and a substrate for  -Gal 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  -Gal under gratuitous conditions p =  (amount of  -Gal)/  (total cell protein)

13. lac system: transcription regulation

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

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

16. 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 2 1

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

19. The PaJaMo experiment Hfr lacI + lacZ + Str S TsX S x F - lacI - lacZ - Str R Tsx R Set a cross in the absence of inducer: After some time, kill the donor with Str and T6 Monitor  -Gal in the presence or in the absence of inducer

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

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

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

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

27. The LAC OPERON

28. LacI binds DNA as a tetramer to better repress transcription Why did Jacob & Monod not find O 2 and O 3 ?

29. X-gal White Blue White Blue White Genetic analysis of the LacI binding sites O 3 O 1 O 2 P lacZ Repression 1300 1.0 1.0 1.0 O 3 O 1 O 2 O 3 O 1 O 2 O 3 O 1 O 2 440 O 3 O 1 O 2 700 O 3 O 1 O 2 1.9 O 3 O 1 O 2 18 O 3 O 1 O 2

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

31. glucose energy glycerol pgi 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) Catabolism ???

32. 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.

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

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

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

36. The lac control region contains three critical cis-acting sites CAP RNAPLacI RNAP

37. lac operator: the regulatory region

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

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

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

41. Different types of negative and positive control of transcription

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

43. -35 -10 15-17 bp RNAP -35 -10 19 bp merT Average Prom. Mechanism of activation by MerR MerR merT -35 -10 Hg ++ MerR 17 bp RNAP

44. 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

45. Crossfeeding analysis of Trp mutants allows to analyze the biochemistry of Trp biosynthesis pathway precursorTrp TrpETrpDTrpB

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

47. the trp attenuator region

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

51. The repressor idea The existence of c and vir mutants. are immune to c, but not vir Immunity of lysogens to superinfection with wt Zygotic induction