slide1 n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Chapter 8 Gene Regulation in Prokaryotes PowerPoint Presentation
Download Presentation
Chapter 8 Gene Regulation in Prokaryotes

Loading in 2 Seconds...

play fullscreen
1 / 142

Chapter 8 Gene Regulation in Prokaryotes - PowerPoint PPT Presentation


  • 460 Views
  • Uploaded on

Chapter 8 Gene Regulation in Prokaryotes. TOPIC 1 Principles of Transcriptional Regulation TOPIC 2 Regulation of Transcription Initiation: Examples from Bacteria TOPIC 3 The Case of Phage λ: Layers of Regulation. TOPIC 1 Principles of Transcriptional Regulation.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

Chapter 8 Gene Regulation in Prokaryotes


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
    Presentation Transcript
    1. Chapter 8 Gene Regulation in Prokaryotes TOPIC 1 Principles of Transcriptional Regulation TOPIC 2 Regulation of Transcription Initiation: Examples from Bacteria TOPIC 3 The Case of Phage λ: Layers of Regulation

    2. TOPIC 1 Principles of Transcriptional Regulation • 1. Gene Expression is Controlled by Regulatory Proteins (调控蛋白) • 2. Most activators and repressors act at the level of transcription initiation • 3. Targeting promoter binding • 4 Targeting transition to the open complex: Allostery regulation (异构调控) after the RNA Polymerase Binding • 5. Action at a Distance and DNA Looping. • 6. Cooperative binding (recruitment) and allostery have many roles in gene regulation

    3. TOPIC 1 Principles of Transcriptional 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) 3

    4. 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

    5. 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. 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.

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

    7. Elongation and termination Elongation Termination

    8. 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. 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. 3. Targeting promoter binding:

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

    10. 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. 4Targeting transition to the open complex: Allostery regulation (异构调控) after the RNA Polymerase Binding

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

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

    13. 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.

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

    15. 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).

    16. Topic 2: Regulation of Transcription Initiation : Examples from Bacteria • Operon: a unit of prokarytoic gene expression and regulation which typically includes: • Structural genes for enzymes in a specific biosynthetic and metabolic pathway whose expression is coordinately controlled. • Control elements, such as operator sequence. • Regulator gene(s) whose products recognize the control elements. These genes is usually transcribed from a different promoter.

    17. The structure of operon

    18. Control element Structural genes

    19. First example: Lac operon 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 or Medicine 1965.

    20. E. coli’s lac operon • E. coli expresses genes for glucose metabolism continuously. • Metabolism of other alternative types of sugars (e.g., lactose) are regulated specifically. • Lactose = disaccharide (glucose + galactose), provides energy. • Lactose acts as an inducer (effector molecule) and stimulates expression of three proteins at 1000-fold increase: • lacZcodes for-galactosidase (半乳糖苷酶) • An enzyme responsible for hydrolysis of lactose to galactose and glucose . • lacYcodes forlactosePermease (半乳糖苷渗透酶) • An enzyme responsible for lactose transport across the bacterial cell wall. • lacAencodes a thiogalactoside transacetylase (硫代半乳糖苷转乙酰酶)to get rid of the toxic thiogalacosides.

    21. -galactosidase and structure of lactose E. Coli cells need an enzyme to break the lactose down into its two component sugars: galactose and glucose. The enzyme that cuts it in half is called  -galactosidase.

    22. -galactosidase and structure of lactose

    23. Structure of the lac operon Lac operon Acetylase -galactosidase Permease DNA lacI: promoter-lacI-terminator operon: promoter-operator-lacZ-lacY-lacA-terminator

    24. -3 — +21 -47 — -84 -47 — -8 -54 —-58 -65 —-69 AUG:+39 — +41

    25. Without inducer-no structure genes expression Regulation genes Lac operon -galactosidase Permease Acetylase DNA transcript No structure genes expression mRNA of repressor translate Inactive lac repressor

    26. Binding of inducer inactivates the lac repressor

    27. cAMP receptor protein The Plac promoter is not a strong promoter. Plac and related promoters do not have strong -35 sequences and some even have weak -10 consensus sequences. For high level transcription, they require the activity of a specific activator protein called cAMP receptor protein (CRP). CRP may also be called catabolite activator protein or CAP. Glucose reduces the level of cAMP in the cell. When glucose is absent, the levels of cAMP in E. coli increase and CRP binds to cAMP.So, the CRP-cAMP complex binds to the lactose operon.

    28. DNA-bending and Transcription regulation CAP-cAMP binding to the lac activator-binding site recruits RNA polymerase to the adjacent lac promoter to form a closed promoter complex. This closed complex then converts to an open promoter complex. CAP-cAMP bends its target DNA by about 90° when it binds. And this is believed to enhance RNA polymerase binding to the promoter, enhancing transcription by 50-fold.

    29. 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

    30. 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.

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

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

    33. Point 3: The mechanism of the binding of regulatory proteins to their sites 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 Olac overlaps promoter (Plac). 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.

    34. The LAC operon

    35. 5. CAP interacts with the CTD domain of the a-subunit of RNAP 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. a CTD: C-terminal domain of the a subunit of RNAP

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

    37. 6. CAP and Lac repressor bind DNA using a common structural motif: helix-turn-helix motif 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.

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

    39. DNA looping Lac operon contains three operators: the primary operator and two other operators located 400 bp downstream and 90 bp upstream. Lac repressor binds as a tetramer (四聚体), with each operator is contacted by a repressor dimer (二聚体). respectively.

    40. 7: Combinatorial Control (组合调控): CAP controls other genes as well. A regulator (CAP) works together with different repressors 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.

    41. Alternative s factor Alternative s factors (可变s因子) direct RNA polymerase to alternative promoters.

    42. factor subunit bound to RNA polymerase for transcription initiation Fig. s and a subunits recruit RNA pol core enzyme to the promoter

    43. E. coli: Heat shock 32 Bacteriophage σ factors Sporulation in Bacillus subtilis Different  factors binding to the same RNAP, conferring each of them a new promoter specificity. 70factors is the most common one in E. coli under the normal growth condition. Many bacteria produce alternative sets of σfactors to meet the regulation requirements of transcription under normal and extreme growth condition. Bacteriophage has its own σfactors

    44. Heat shock (热休克) Around 17 proteins are specifically expressed in E. coli when the temperature is increased above 37ºC. These proteins are expressed through transcription by RNA polymerase using an alternative  factor 32 coded by rhoH gene. 32has its own specific promoter consensus sequences.

    45. Bacteriophages Many bacteriophages synthesize their own σfactors to endow the host RNA polymerase with a different promoter specificity and hence to selectively express their own phage genes .

    46. B. subtilis SPO1 phage expresses a cascade of σfactors which allow a defined sequence of expression of different phage genes.

    47. Third example: NtrC and MerR use allosteric activation Transcriptional activators NtrC and MerR work by allostery rather than by recruitment The majority of activators work by recruitment, such as CAP. These activators simply bring an active form of RNA polymerase to the promoter. The beautiful exceptions: allosteric activation by NtrC and MerR. In allosteric activation RNAP initially binds the promoter in an inactive complex, and the activator triggers an allosteric change in that complex to activate transcription.

    48. 1. NtrC has ATPase activity and works at DNA sites far away from the gene. NtrC controls expression of genes involved in nitrogen metabolism (氮代谢), such as the glnA gene. NtrC has separate activating and DNA-binding domains, and binds DNA only when the nitrogen levels are low.

    49. Low nitrogen levels (低水平氮)NtrC phosphorylation and conformational change NtrC (?) binds DNA sites at ~-150 bp position as a dimer (?)NtrC interacts 54 in RNAP bound to the glnA promoter  NtrCATPase activity provides energy needed to induce a conformation change in RNAP transcription STARTs activation by NtrC