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Chapter 12 Gene Regulation in Prokaryotes

Chapter 12 Gene Regulation in Prokaryotes. 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.

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Chapter 12 Gene Regulation in Prokaryotes

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  1. Chapter 12Gene 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: • Are expressed at a fixed rate, irrespective to the cell condition. • They are essential for basic processes involving in cell replication and growth • Controllablegenes: • 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

  4. 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: • Mutations in protein-coding gene sequences. • Mutations in regulatory sequences.

  5. The 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)

  6. 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

  7. 2. Most activators and repressors act at the level of transcription initiation 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.

  8. 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.

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

  10. Elongation Termination

  11. 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.

  12. Generally, RNAP binds many promoters weakly. Why? Activatorscontain 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 calledrecruitment regulation (招募调控).*** On the contrary, Repressorscan bind to the operator inside of the promoter region, which prevents RNAP binding and the transcription of the target gene.

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

  14. 4Targeting 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.

  15. 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.

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

  17. 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.

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

  19. 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) : produce sensitive switches to rapidly turn on a gene expression. (1+1>2) integrate signals (some genes are activated when multiple signals are present).

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

  21. 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

  22. General structure of an OPERON

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

  24. Point 1: Composition of the Lacoperon

  25. 1. Lactose operon contains3 structural genes and 2 control elements. 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

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

  27. 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

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

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

  30. 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 31 The LAC operon

  31. 3. The activity of Lac repressor and CAP are controlled allosterically by their signals. Allolactose binding: turn off 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

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

  33. 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

  34. Regulation of lac Operon Expression Off Off

  35. Functional state of the E. colilac operon in the absence of lactose:

  36. Functional state of the E. colilac operon growing on lactose:

  37. Positive control of the lac operon with CAP

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

  39. 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). 40 The LAC operon

  40. 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.

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

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

  43. Lactose/allolactose is a native inducer to release RNA transcription from Plac. 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.

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

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

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

  47. 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.

  48. 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 structure Signal • Paired region 1-2 pause • Paired region 2-3 anti-termination • Paired region 3-4 termination

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

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