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Welcome Each of You to My Molecular Biology Class

Welcome Each of You to My Molecular Biology Class. 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. Part IV Regulation.

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Welcome Each of You to My Molecular Biology Class

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  1. Welcome Each of You to My Molecular Biology 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. Part IV Regulation Ch 16: Regulation in prokaryotes Ch 17: Regulation in eukaryotes Ch 18: Regulation during development Ch 19: Comparative genomics and evolution of animal diversity

  4. 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.

  5. Chapter 16 Regulation principles and How genes are regulated in bacteria Chapter 17 Basic mechanism of gene expression in eukaryotes Chapter 18 How genes are regulated to bestow cell type specificity in a group of genetically identical cells Chapter 19 How different animals diverse in genomes, why human is so special?

  6. Surfing the contents of Part IV

  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 Examples of Gene Regulation after Transcription Initiation (Trp operon) TOPIC 4 The Case of Phage λ: Layers of Regulation

  10. CHAPTER 16 Gene Regulation in Prokaryotes Topic 1: Principles of Transcription Regulation 5/10/2005

  11. 1-1 Gene Expression is Controlled by Regulatory Proteins 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. What are the transcription steps targeted by the regulators ?

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

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

  15. Principles of Transcription Regulation 1-2 Targeting promoter binding: Many promoters are regulated by activators that help RNAP bind DNA and by repressors that block the binding • At many promoters, RNAP binds weakly • Lac operon is a good example

  16. Fig 16-1 a. Absence of Regulatory Proteins (operator) b. To Control Expression c. To Activate Expression

  17. 1-3 Targeting transition to the open complex: Some Activators Work by Allostery and Regulate Steps after RNA Polymerase Binding Fig 16-2 Examples: Activator promoter NtrC glnA MerR merT

  18. Some promoters are inefficient at more than one step and can be activated by more than one mechanism Repressors can work in ways other than just blocking the promoter binding. For example, inhibition of the transition to the open complex.

  19. Principles of Transcription Regulation 1-6 Targeting termination and beyond: Antitermination and Beyond • The bulk of gene regulation takes place at the initiation of transcription. • Some involve transcriptional elongation/termination, RNA processing, and translation of the mRNA into protein.

  20. 1-4 Action at a Distance and DNA Looping. Some proteins interact with each other even when bound to sites well separated on the DNA Fig 16-3

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

  22. 1-5 Cooperative Binding and Allostery have Many Roles in Gene Regulation • Cooperative binding: the activator interacts simultaneously with DNA and polymerase and so recruits the enzyme to the promoter • Group of regulators often bind DNA cooperatively: (1) produce sensitive switches to rapidly turn on a gene expression, (2) integrate signals (some genes are activated when multiple signals are present)

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

  24. CHAPTER 16 Gene Regulation in Prokaryotes Topic 2: Regulation of Transcription Initiation : Examples from Bacteria 5/10/2005

  25. Operon:a unit of prokarytoic gene expression and regulation which typically includes: 1.Structural genesfor enzymes in a specific biosynthetic pathway whose expression is coordinately controlled. 2.Control elements, such as operator sequence. 3.Regulator gene(s)whose products recognize the control elements. Sometimes are encoded by the gene under the control of a different promoter

  26. Control element Structural genes

  27. Regulation of Transcription Initiation in Bacteria First example: Lac operon 5/10/2005

  28. The lactose (Lac) Operon (乳糖操纵子) Fig 16-5 The enzymes required for the use of lactose as a carbon source are only synthesized when lactose is available as the sole carbon source. The LAC operon

  29. Lactose operon: a regulatory gene and 3 stuctural genes, and 2 control elements Regulatory gene Structural Genes Cis-acting elements DNA lacI lacZ lacY lacA PlacI Olac Plac m-RNA Protein Transacetylase β-Galactosidase Permease The LAC operon

  30. 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

  31. 1. The lacZ, lacY, lacA genes are transcribed into a single lacZYA mRNA (called polycistronic message) under the control of a signal promoter Plac. 2. 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

  32. 2-1: An activator and a repressor together control the lac genes The activator: CAP (Catabolite Activator Protein) or CRP (cAMP Receptor Protein); responses to the glucose level. The repressor: lac repressor that is encoded by LacIgene; responses to the lactose. The LAC operon

  33. The LAC operon Fig 16-6

  34. The LAC operon 2-2: CAP and lac repressor have opposing effects on RNA polymerase binding to the lac promoter

  35. The LAC operon The site bound by lac repressor is called the lac operator.

  36. The LAC operon Fig 16-8 The lac operatoroverlaps promoter, and so repressor bound to the operator physically prevents RNA polymerase from binding to the promoter.

  37. CAP binds to a site with the similar structure as the operator, which is 60 bp upstream of the start site of transcription. • CAP also interacts with the enzyme and recruit it to the promoter. Fig 16-9 a CTD: C-terminal domain of the a subunit of RNAP

  38. The LAC operon 2-3: CAP has separate activating and DNA- binding surface CAP binds as a dimer a CTD Fig 16-10

  39. The LAC operon 2-4: CAP and lac repressor bind DNA using a common structural motif

  40. Both CAP and lac repressor bind DNA using a helix-turn-helix motif. One is the recognition helix that can fits into the major groove of the DNA. Fig 16-11 The LAC operon

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

  42. Lac repressor binds as a tetramer, with each operator is contacted by a repressor dimer. In addition to the primary operator, there are two other lac operators located 400 bp downstream and 90 bp upstream, respectively. Fig 16-13 Not all the binding use ahelix-turn-helix motif

  43. 2-5: The activity of Lac repressor and CAP are controlled allosterically by their signals Binding of the corresponding signals alter the structure of these two regulatory proteins The LAC operon

  44. Response to lactose Absence of lactose z y a i p o Active Very low level of lac mRNA Lack of inducer: the lac repressor block all but a very low level of trans-cription of lacZYA . Lactose is present, the low basal level of permease allows its uptake, andβ-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

  45. Response to glucose The LAC operon

  46. Regulation of Transcription Initiation in Bacteria • A regulator (CAP) works together with different repressor at different genes, this is an example of Combinatorial Control. • In fact, CAP acts at more than 100 genes in E.coli, working with an array of partners. 2-6: Combinatorial Control (组合调控): CAP controls other genes as well

  47. Regulation of Transcription Initiation in Bacteria Second example: Alternative s factor 5/10/2005

  48. Alternative s factors 2-7: Alternative s factor direct RNA polymerase to alternative site of promoters

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