1. Chapter 10 The Operon Tsung-Luo Jinn

2. Gene expression controlled at: Transcription—initiation,termination, not elongation Processing—in EuK RNA splicing, modification, transporting translation -- in ProK coupled transcription and translation Translation--like transcription

3. Genes products of: Structural genes: functions as: structure proteins, enzymes regulatory proteins Regulatory genes: functions: a specific protein in regulation of gene expression, by binding to particular sites on DNA *cistrons *constitutive expression!!

4. Gene regulatory mechanism : Negative control: Repressor binds to operator, gene turn off Positive control: Activator binds to operator, gene turn on *inducible repressible

5. Negative control—Bacteria Repressor,Operator **polycistronic mRNA

6. Positive control—ProK, EuK Cis-acting sites, trans-acting factors **monocistronic mRNA

7. Regulator bind to DNA ? Gene Expression Positive regulation Negative regulation YES No ON OFFON OFF

8. Operon model Jacob and Monod, 1961 The control of gene expression by induction and repression How to control gene expression in Bacteria: Two types of DNA sequences: Coding for protein—trans-acting proteins Non-coding—cis-acting sequences ** gene expression is controlled by: Interaction of the trans-factor with cis-element

9. Topics: Regulation of Gene Expression Metabolic regulation—Lac operone Catabolite repression Stringent response Attenuation—Trp operon

10. Gene Regulation Negative control Positive control

11. Metabolic regulation lec operon The

13. The lac operon controls utilization of Lactose Coding region Control region Regulatory gene operon 4er dimer

15. How lac I gene control lac ZYA gene transcription ? Negative regulation-- lac genes are transcribed unless it was turned off by a repressor protein Repressor–-lac I product Inducer—lactose (allolactose), IPTG *isopropyl thioglactoside

16. Induction of gene expression in lac operon Inducers

17. Induction of lac genes expression --- by inducer Induceable enzyme— From ~ 5 to 5,000 molecular Shot life of mRNA— Half-life ~3 min Stable of enzyme * Is de novo synthesis !!! Gratuitous inducer—IPTG

18. Mutations: O-type and I-type Repressor: I - - -loss of function mutant --Constitutive expression --trnas-dominant Opertor: O c --Constitutive expression -- Cis-dominant LacI I S - -abolish the ability of bind to inducer --Uninducible I -d - -loss of function to bind the operator --Constitutive expression

19. I - Z + / I + Z + O + Z + / O C Z + O C Z + / I - Z + I + Z + / I - Z + O C Z - / O + Z + O C Z + / O + Z + O C Z + / O + Z - Gene typesConstitutive Inducible - + + + + - - - - - - + + + *Interallelic complementation Conclusions

20. Map of mutation sites

21. The operator is palindromic—with inverted repeats TGTGTG--------------------- ---------------------TGTGTG

22. Two models for repressor action

23. The crystal structure of a monomer of Lac repressor HTH headpiece core C-terminal Oligomeriation Inducer biding Operator biding

26. i p z O3O3 O2O2 CAP O1O1 Three operator sequences O2O2 O1O1 O3O3 : the original operator : 410 bp downstream in lacZ : 83 bp upstream in lacI dimer * This structure enhanced RNA polrmerase binding (100x)/store at promoter Repressor tetramer binds to

27. The trp repressor controls three unlinked sets of genes * Autogenous control

28. Operators lie on different positions relative to promoter

29. Catabolite repression

30. Medium with Glucose and Lactose— It metabolizes glucose and represses the use of lactose—Catabolite repression * How??

31. ATP cAMP Adenyl cyclase Glucose × CAP cAMP-CAP complex Gene turn on

32. Positive control of gene expression *mutations: I -,O c CAP: a product of Cap gene No lac mRNA cyclase: a product of Cya gene promoter Cya -, Cap -

33. The CAP dimer binding site with a conserved TGTAG pentamer

34. CAP biding to different region relative to promoter

36. CAP cause DNA to bend

37. The Stringent Response Adverse growth condition induce

38. The Stringent Response Massive reduction in the synthesis of rRNA and tRNA (10-20X) Reduced mRNA synthesis (~3X) Increase protein degradsation Reduce synthesis of nucleotides, carbonhydrates and lipids, ect Amino acid starvation Accumulation o ppGpp and pppGpp nucleotides

39. The synthesis of ppGpp under Amino acid starvation Function: is to bind to target proteins to alter their activity Trigger :uncharged tRNA in the A site of ribosome

40. P A P A

41. What does ppGpp do? Initiation is specifically inhibited at the promoters of operons coding for rRNA The elongation of transcription of many or MOST template is reduced *ppGpp is a broad spectrum inhibitors

42. Autogenous control at translation level

44. Attenuation of gene expression The Trp operon

45. Attenuation : a mechenism that controls the ability of RNA polymerase to read through an attenuator, which is an intrinsic terminator located at the beginning of a transcription unit. The changes in secondary structure that control attenuation are determined by the position of the ribosome on mRNA. The external circumstances is influenced ribosome movement in the leading sequence of mRNA.

46. The trp operon Repressor: trpR gene coded in different locus （ inactive ） Corepressor : trytophane With two different separate mechanism Attenuation control

48. The trp operon—leader sequence Chorismic acid to tryptophane 11 12

50. The structure on the leader region W/ trp W/O trp 1 23 4 12 34

51. 123 4 21 34 tRNA trp 【 Trp 】 （ ）

52. Antisense RNA regulates transcription/translation Prevent initiation of protein synthesis Destabilization of dRNA by endonuclease Premature termination of transcription

54. RNAi PNAS (2,000) 97:4885-4990