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Welcome to MB Class

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  1. Welcome to MB Class

  2. Molecular Biology of the Gene, 5/E--- Watson et al. (2004) Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods 2005-5-10

  3. The revised central dogma The structure of DNA and RNA 基因组的保持 基因组的表达 RNA processing Gene regulation

  4. Part IV Regulation Ch 16: Transcriptional regulation in prokaryotes Ch 17: Transcriptional regulation in eukaryotes Ch18: Regulatory RNAs Ch 19: Gene regulation in development and evolution Ch 20: Genome Analysis and Systems Biology

  5. Expression of many genes in cells are regulated Housekeeping genes: expressed constitutively, essential for basic processes involving in cell replication and growth. Inducible genes: expressed only when they are activated by inducers or cellular factors.

  6. Surfing the contents of Part IV --The heart of the frontier biological disciplines

  7. Some of the peoples who significantly contribute to the knowledge of gene regulation

  8. Chapter 16 Gene Regulation in Prokaryotes • Molecular Biology Course

  9. TOPIC 1Principles of Transcriptional Regulation [watch the animation] TOPIC 2 Regulation of Transcription Initiation: Examples from Bacteria (Lac operon, alternative s factors, NtrC,MerR, Gal rep, araBAD operon) TOPIC 3 The Case of Phage λ: Layers of Regulation

  10. CHAPTER 16 Gene Regulation in Prokaryotes Topic 1: Principles of Transcription Regulation 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)

  11. 1. Gene Expression is Controlled by Regulatory Proteins (调控蛋白) Principles of Transcription Regulation 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

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

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

  14. Fig 12-3-initiation Promoter Binding (closed complex) Promoter “melting” (open complex) Promoter escape/Initial transcription

  15. Fig 12-3-Elongation and termination Elongation Termination

  16. 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. Principles of Transcription Regulation

  17. 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.

  18. Fig 16-1 a. Absence of Regulatory Proteins: basal level expression b. Repressor binding to the operator represses expression c. Activator binding activates expression

  19. 4Targeting transition to the open complex:Allostery regulation (异构调控)after the RNA Polymerase Binding Principles of Transcription Regulation 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.

  20. Allostery regulation Fig 16-2 Allostery is not only a mechanism of gene activation , it is also often the way that regulators are controlled by their specific signals.

  21. Repressors can work in ways: blocking the promoter binding. blocking the transition to the open complex. blocking promoter escape

  22. 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. Principles of Transcription Regulation Fig 16-3

  23. Fig 16-4 DNA-binding protein can facilitate interaction between DNA-binding proteins at a distance Fig 16-4 Architectural protein

  24. 6. Cooperative binding (recruitment) and allostery have many roles in gene regulation Principles of Transcription 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) : produce sensitive switches to rapidly turn on a gene expression. (1+1>2) integrate signals (some genes are activated when multiple signals are present).

  25. Watch the animation-regulation of the transcription initiation!

  26. CHAPTER 16 Gene Regulation in Prokaryotes Topic 2: Regulation of Transcription Initiation : Examples from Bacteria

  27. Operon:a unit of prokarytoic gene expression and regulation which typically includes: 1.Structural genesfor enzymes in a specific biosynthetic and metabolic pathway whose expression is coordinately controlled. 2.Control elements, such as operator sequence. 3.Regulator gene(s)whose products recognize the control elements. These genes is usually transcribed from a different promoter.

  28. Control element Structural genes

  29. Regulation of Transcription Initiation in Bacteria First example: Lac operon The lactose Operon (乳糖操纵子)

  30. Point 1: Composition of the Lacoperon

  31. 1. Lactose operon contains3 structural genes and 2 control elements. Fig 16-5 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

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

  33. The lacZ, lacY, lacA genes are transcribed into a single lacZYA mRNA (polycistronic mRNA) under the control of a single promoter Plac. LacZYA transcription unit contains anoperator site Olac position between bases -5 and +21 at the 3’-end of Plac Binds with the lac repressor The LAC operon

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

  35. 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 LacIgene; responses to the lactose. Sugar switch-off mechanism The LAC operon

  36. 3. The activity of Lac repressor and CAP are controlled allosterically by their signals. Allolactose binding: turn of Lac repressor cAMP binding: turn on CAP 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

  37. The LAC operon Fig 16-6

  38. Absence of lactose z y a i p o Active Very low level of lac mRNA Response to lactose Lack of inducer: the lac repressor block all but a very low level of trans-cription of lacZYA . When Lactose is present, the low basal level of permease allows its uptake, and b-galactosidase catalyzes the conversion of some lactose to allolactose. Allolactoseacts as an inducer, binding to the lac repressor and inactivate it. Presence of lactose z y a i p o Inactive Permease Transacetylase b-Galactosidase

  39. Response to glucose: 注意该图的CRP结合位点有误

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

  41. The LAC operon 4. CAP and Lac repressor have opposing effects on RNA polymerase 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 (Olac), and the Olacoverlaps promoter (Plac). The LAC operon

  42. The LAC operon 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 Plac. The LAC operon

  43. The LAC operon Fig 16-8

  44. The LAC operon 5. CAP interacts with the CTD domain of the a-subunit of RNAP The LAC operon

  45. CAPsite has the similar structure as the operator, which is 60 bp upstream of the start site of transcription. • CAP interacts with the CTD domain of the a-subunit of RNAP and thus promotes the promoter binding by RNAP. Fig 16-9 a CTD: C-terminal domain of the a subunit of RNAP

  46. The LAC operon CAP binds as a dimer a CTD Fig 16-10. CAP has separate activating and DNA-binding surface

  47. 6. CAP and Lac repressor bind DNA using a common structural motif: helix-turn-helix motif Fig 16-11 One is the recognition helix that can fits into the major groove of the DNA. Another one sits across the major grove and makes contact with the DNA backbone. The LAC operon

  48. DNA binding by a helix-turn-helix motif Fig 16-12 Hydrogen Bonds between l repressor and the major groove of the operator.