1. Chapter 12 Gene Regulation in Prokaryotes

2. Gene Regulation Is Necessary? By switching genes off when they are not needed, cells can prevent resources from being wasted. There should be natural selection favoring the ability to switch genes on and off. Complex multicellular organisms are produced by cells that switch genes on and off during development. A typical human cell normally expresses about 3% to 5% of its genes at any given time. Cancer results from genes that do not turn off properly. Cancer cells have lost their ability to regulate mitosis, resulting in uncontrolled cell division

3. Classification of gene with respect to their Expression Constitutive ( house keeping) genes: Constitutive ( house keeping) genes: – Are expressed at a fixed rate, irrespective to the cell condition. – They are essential for basic processes involving in cell replication and growth Controllable genes: Controllable genes: – Are expressed only as needed. Their amount may increase or decrease with respect to their basal level in different condition. – Their structure is relatively complicated with some response elements

5. Regulation of gene expression lac operon was the first discovered example of a gene regulation system by Francois Jacob and Jacques Monod (Pasteur Institute, Paris, France) Studied the organization and control of the lac operon in E. coli. Earned Nobel Prize in Physiology / Medicine 1965. Studied 2 different types of mutations in the lac operon: 1.Mutations in protein-coding gene sequences. 2.Mutations in regulatory sequences.

6. What are the regulatory proteins? Which steps of gene expression to be targeted? How to regulate? (recruitment, allostery, blocking, action at a distance, cooperative binding) The Principles of Transcription Regulation

7. 1. Gene Expression is Controlled by Regulatory Proteins ( 调控蛋白 ) Gene expression is very often controlled by Extracellular Signals, which are communicated to genes by regulatory proteins:  Positive regulators or activators INCREASE the transcription  Negative regulators or repressors DECREASE or ELIMINATE the transcription

8. 2. Most activators and repressors act at the level of transcription initiation Why that? 1.Transcription initiation is the most energetically efficient step to regulate. [A wise decision at the beginning] 2.Regulation at this step is easier to do well than regulation of the translation initiation.

9. Regulation also occurs at all stages after transcription initiation. Why? 1.Allows more inputs and multiple checkpoints.. 2. The regulation at later stages allow a quicker response.

10. Promoter Binding (closed complex) Promoter “melting” (open complex) Promoter escape/Initial transcription

11. Termination Elongation

12. 12 recruitment block 3. Targeting promoter binding: Many promoters are regulated by activators ( 激活蛋白 ) that help RNAP bind DNA (recruitment) and by repressors ( 阻遏蛋白 ) that block the binding.

13. Generally, RNAP binds many promoters weakly. Why? Activators contain two binding sites to bind a DNA sequence and RNAP simultaneously, can therefore enhance the RNAP affinity with the promoters and increases gene transcription. This is called recruitment regulation ( 招募调控 ).*** On the contrary, Repressors can bind to the operator inside of the promoter region, which prevents RNAP binding and the transcription of the target gene.

14. 14 a. Absence of Regulatory Proteins: basal level expression b. Repressor binding to the operator represses expression c. Activator binding activates expression

15. 4 Targeting transition to the open complex: Allostery regulation ( 异构调控 ) after the RNA Polymerase Binding In some cases, RNAP binds the promoters efficiently, but no spontaneous isomerization ( 异构化 ) occurs to lead to the open complex, resulting in no or low transcription. Some activators can bind to the closed complex, inducing conformational change in either RNAP or DNA promoter, which converts the closed complex to open complex and thus promotes the transcription. This is an example of allostery regulation.

16. Allostery regulation Allostery is not only a mechanism of gene activation, it is also often the way that regulators are controlled by their specific signals.

17. Repressors Repressors can work in ways: (1) blocking the promoter binding. (2) blocking the transition to the open complex.

18. 18 5. Action at a Distance and DNA Looping. The regulator proteins can function even binding at a DNA site far away from the promoter region, through protein-protein interaction and DNA looping.

19. DNA-binding protein can facilitate interaction between DNA-binding proteins at a distance Architectural protein

20. 20 6. Cooperative binding (recruitment) and allostery have many roles in gene regulation For example: group of regulators often bind DNA cooperatively (activators and/or repressors interact with each other and with the DNA, helping each other to bind near a gene they regulated) : (1) produce sensitive switches to rapidly turn on a gene expression. (1+1>2) (2) integrate signals (some genes are activated when multiple signals are present).

21. Topic 2: Regulation of Transcription Initiation : Examples from Bacteria

22. OPERON in gene regulation of prokaryotes Definition: a cluster of genes in which expression is regulated by operator-repressor protein interactions, operator region, and the promoter. Its structure: Each Operon is consisted of few structural genes( cistrons) and some cis-acting element such as promoter (P) and operator (O). Its regulation: There are one or more regulatory gene outside of the Operon that produce trans-acting factors such as repressor or activators. Classification: 1- Catabolic (inducible) such as Lac OPERON 2- Anabolic (repressible) such as ara OPERON 3- Other types

23. General structure of an OPERON

24. First example: Lac operon The lactose Operon ( 乳糖操纵子 )

25. Point 1: Composition of the Lac operon

26. The enzymes encoded by lacZ, lacY, lacA are required for the use of lactose as a carbon source. These genes are only transcribed at a high level when lactose is available as the sole carbon source. The LAC operon 1. Lactose operon contains 3 structural genes and 2 control elements.

27. lacZ lacY lacA codes for β -galactosidase ( 半乳 糖苷酶 ) for lactose hydrolysis encodes a cell membrane protein called lactose permease ( 半乳糖苷 渗透酶 ) to transport Lactose across the cell wall encodes a thiogalactoside transacetylase ( 硫代半乳糖苷转乙 酰酶 ) to get rid of the toxic thiogalacosides

28. polycistronic mRNA The lacZ, lacY, lacA genes are transcribed into a single lacZYA mRNA (polycistronic mRNA) under the control of a single promoter P lac. LacZYA transcription unit contains an operator site O lac position between bases -5 and +21 at the 3’-end of P lac Binds with the lac repressor

29. i Operon Regulatory Gene Plac Olacz y a DNA m-RNA β -Galactosidase Permease Transacetylase Protein Control elements -5 +21 repressor

30. 30 Point 2: Regulatory proteins and their response to extracellular signals

31. 31 2. An activator and a repressor together control the Lac operon expression The activator: CAP (Catabolite Activator Protein, 代谢产物激活蛋白 ) or CRP (cAMP Receptor Protein, cAMP 受体蛋白 ); responses to the glucose level. The repressor: lac repressor that is encoded by LacI gene; responses to the lactose. Sugar switch-off mechanism The LAC operon

32. 3. The activity of Lac repressor and CAP are controlled allosterically by their signals. Lactose is converted to allolactose by b-galactosidase, therefore lactose can indirectly turn off the repressor. Glucose lowers the cellular cAMP level, therefore, glucose indirectly turn off CAP. The LAC operon Allolactose binding: turn off Lac repressor cAMP binding: turn on CAP

33. Lac OPERON an inducible Operon In the absence of lac In the presence of lac

34. CRP or CAP is positive regulator of Lac and some other catabolic Operons CRP= Catabolic gene regulatory Protein CRP= cAMP receptor Protein CAP= Catabolic gene Activating Protein

35. Regulation of lac Operon Expression Off

36. Functional state of the E. coli lac operon in the absence of lactose:

37. Functional state of the E. coli lac operon growing on lactose:

38. Positive control of the lac operon with CAP

39. 39 Point 3: The mechanism of the binding of regulatory proteins to their sites

40. 40 Repressor binding physically prevents RNAP from binding to the promoter Repressor binding physically prevents RNAP from binding to the promoter, because the site bound by lac repressor is called the lac operator (O lac ), and the O lac overlaps promoter (P lac ). 4. CAP and Lac repressor have opposing effects on RNA polymerase binding to the promoter The LAC operon

41. CAP binds to a site upstream of the promoter, and helps RNA polymerase binds to the promoter by physically interacting with RNAP. CAP binds to a site upstream of the promoter, and helps RNA polymerase binds to the promoter by physically interacting with RNAP. This cooperative binding stabilizes the binding of polymerase to P lac.

42. Base pair sequence of controlling sites, promoter, and operator for lac operon of E. coli.

43. 43 CAP interacts with the CTD domain of the a-subunit of RNAP and thus promotes the promoter binding by RNAP  CTD: C-terminal domain of the a subunit of RNAP 5. CAP interacts with the CTD domain of the a-subunit of RNAP

44. Lactose/allolactose is a native inducer to release RNA transcription from P lac. IPTG (isopropyl-  -D-thiogalacto- pyranoside, 异丙基 -β-D- 硫代吡喃半乳糖 苷 ), a synthetic inducer, can rapidly stimulate transcription of the lac operon structural genes.  IPTG is used to induce the expression of the cloned gene from lac promoter in many vectors, such as pUC19.

45. Back Amp r ori pUC18 (3 kb) MCS (Multiple cloning sites, 多克隆位点） Lac promoter lacZ’ Gene X No IPTG, little protein X With IPTG, a lot of protein X

46. 46 Second example: The Trp operon of E. coli

47. Trp OPERON a repressible example In the absence of Trp In the presence of Trp

48. Regulation of the trp operon: 1. Repressor/operator interaction – When tryptophan is present, tryptophan binds to trpR gene product. – trpR protein binds to the trp operator and can only bind to the operator, thus prevents transcription. – Repression reduces transcription of the trp operon ~70- fold.

49. 2. Molecular model for attenuation( 弱化作用 ） : Recall that a leader region (trpL) occurs between the operator and the trpE sequence. Within this leader is the attenuator sequence (att). att sequence contains a start codon, 2 Trp codons, a stop codon, and four regions of sequence that can form three alternative secondary structures. Secondary structureSignal Paired region 1-2pause Paired region 2-3anti-termination Paired region 3-4termination

50. Organization of the leader/attenuator trp operon sequence.

51. Attenuation model in Trp starved cells

52. Molecular model for attenuation (cont.): Position of the ribosome plays an important role in attenuation: When Trp is scarce or in short supply (and required): 1.Trp-tRNAs are unavailable, ribosome stalls at Trp codons and covers attenuator region 1. 2.Region 1 cannot pair with region 2, instead region 2 pairs with region 3 when it is synthesized. 3.Region 3 (now paired with region 2) is unable to pair with region 4 when it is synthesized. 4.RNA polymerase continues transcribing region 4 and beyond synthesizing a complete trp mRNA.

53. Attenuation model in Trp non-starved cells

54. Molecular model for attenuation (cont.): Position of the ribosome plays an important role in attenuation: When Trp is abundant (and not required): 1.Ribosome does not stall at the Trp codons and continues translating the leader polypeptide, ending in region2. 2.Region 2 cannot pair with region 3, instead region 3 pairs with region 4. 3.Pairing of region 3 and 4 is the “attenuator” sequence and acts as a termination signal. 4.Transcription terminates before the trp synthesizing genes are reached.

55. 36 The attenuators of some operons