MB 206 : Module 1 - B

1. Regulation of Gene Expression in Bacteria MB 206 : Module 1 - B Angelia Teo Jan 09

2. This diagram is for eukaryote Angelia Teo Jan 09

3. Regulation of Gene Expression • A cell contains the entire genome of an organism– ALL the DNA. • Gene expression= transcribing and translating the gene • Regulation allows an organism to selectively transcribe (and then translate) only the genes it needs to. • Genes expressed depend on • the type of cell • the particular needs of the cell at that time.

4. Principles of Gene Regulation In genetics, constitutive refers to a gene product made all the time. In the absence of the activator or the repressor, RNA polymerase transcribes the gene constitutively A gene is expressed in higher level under influence of some signal Angelia Teo Jan 09

5. How Are Genes Regulated? • Genes located in coherent packages called operons • operons has 4 parts • regulatory gene - controls timing or rate of transcription • promoter - starting point • operator - controls access to the promoter by RNA polymerase • structural genes • NOTE = operons regulated as units Angelia Teo Jan 09

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7. Gene Regulation in Prokaryotes • Prokaryotes organize their genome into operons • Operon = a group of related genes • One promoter sequence at the very beginning • All of the genes will be transcribed together (in one long strand of RNA.

8. Principle of Gene Regulation • RNA polymerase binds to DNA at promoters. • Transcription initiation is regulated by proteins that bind to or near promoters. • Repression of a repressible gene: (i.e., negative regulation) repressors (vs activitors) bind to operators of DNA. • Repressor is regulated by an effector, usually a small molecules or a protein, that binds and causes a conformational change. • Activitor binds to DNA sites called enhancer to enhance the RNA polymerase activity. (i.e., [positive regulation) • Induction of an inducible gene, e.g., heat-shock genes. Angelia Teo Jan 09

9. General organization of an inducible gene

10. Regulation of Genes Transcription Factor (Protein) RNA polymerase DNA Regulatory Element Gene

11. Regulation of Genes New protein RNA polymerase Transcription Factor DNA Regulatory Element Gene

12. Gene Expression How much protein is in a cell (and active)?? Angelia Teo Jan 09

13. Most genes are not expressed at a particular time • Not all of the genes in a bacteria will be expressed at the same time. • Even in some of the smallest bacteria, about 500 different genes exists • Of the 4279 genes in E. coli , only about 2600 (~60%) are expressed in standard laboratory conditions. • Only about 350 genes are expressed at more than 100 copies (i.e. molecules!) per cell, making up 90% of the total protein. Angelia Teo Jan 09

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16. Possible target in control of gene expression

17. Topics: • Lac Operon(Negative control & Catabolic repression) • Tryptophan Operon (Positive control) • Histidine Operon (Attenuator) Angelia Teo Jan 09

18. Comparison of genomes of various organism Angelia Teo Jan 09

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20. Genes in E.coli Angelia Teo Jan 09

21. E.coli genes expressed • A total of 4288 genes in Escherichia coli - 2600 genes found under standard laboratory growth conditions - 2100 protein spots detected under 2-D protein gels - 350 proteins in high amount, the rest are very low amounts • Majority of the genes are likely to be expressed transiently, in small amounts during DNA replication, and then remain silent (unexpressed) until the next round of DNA synthesis

22. Why is there a need to control gene expression? 1) Prevent energy wastage 2) Ensure only necessary proteins are made according to the requirement for cells growth. • Small portion of DNA in cell used for genetic message (mRNA), the rest for regulatory purposes. Angelia Teo Jan 09

23. Gene Regulation in bacteria • How do single-celled prokaryotes like E. coli know how to respond to their environments? • Each environmental cue generates a specific response, with specific proteins and reactions. eg. A bacterium can use different sources of Nitrogen - incorporate N2 gas from the air - incorporate ammonia from their surroundings or - from amine group of an amino acid like glutamine (easier and less energy) These processes involve very different enzymes. Presence of glutamine, the cell will turn off synthesis of enzymes for fixing N2 Angelia Teo Jan 09

24. How can the cell "turn off" the synthesis of proteins from its DNA? Angelia Teo Jan 09

25. Gene regulation can occur at any place along the flow of information from DNA to RNA to protein: Angelia Teo Jan 09

26. Different forms of gene regulation a. Regulation by DNA Replication (default) b. Transcriptional Regulation by different s-factors. c. Negative Regulation of Gene Expression d. Positive Control of Gene Regulation e. Alternative splicing of RNA (almost exclusively for eukaryotes) f. Post-transcriptional regulation - termination of transcription - translation control - message stability - protein stability Angelia Teo Jan 09

27. E.coli RNA Polymerase subunits Angelia Teo Jan 09

28. Transcription regulation by s-factors • s70 - RpoD “normal” s-factor s54 - RpoN Nitrogen response s38 - RpoS Stationary phase s32 - RpoH Heat shock response s28 - FliA Flagellar genes regulation s24 - RpoE Heat shock high temp. • Approx: 1500 - 2000 copies of RNAP holoenzyme/ cell • For bacteria growing in "log phase": • ~600 copies of RpoD (s70) • ~200 copies of RpoS (s38) • [RpoS] increases to ~600 copies per cell in stationary phase or osmotic shock. Angelia Teo Jan 09

29. Operon and Regulon • An operon - consists of a set of genes expressed coordinately & transcribed as a single unit - Specific regulation (positive / negative) can induce or repress a particular gene or operon - contains both a regulatory & a message region. - Regulatory / control region at the 5’ side of the gene & codes for a protein (message region). • Regulon - comprise of global regulation affecting a set of operons. - All operons in the regulon are coordinately controlled by the same regulatory mechanism. Angelia Teo Jan 09

30. Operons-the basic concept of Prokaryotic Gene Regulation • Regulated genes can be switched on and off depending on the cell’s metabolic needs • Operon : a regulated cluster of adjacent structural genes, operator site, promotor site, and regulatory gene(s)

31. Repressible vs. Inducible Operonstwo types of negative gene regulation Repressible Operons • Genes are initially ON • Anabolic pathways • End product switches off its own production Inducible Operons • Genes are initially OFF • Catabolic pathways • Switched on by nutrient that the pathway uses

32. lac: an inducible operon

33. The lac Operon of E. coli 1. Growth and division genes of bacteria are regulated genes. Their expression is controlled by the needs of the cell as it responds to its environment with the goal of increasing in mass and dividing. 2. Genes that generally are continuously expressed are constitutive genes (housekeeping genes). Examples include protein synthesis and glucose metabolism. 3. All genes are regulated at some level, so that as resources dwindle the cell can respond with a different molecular strategy. 4. Prokaryotic genes are often organized into operons that are cotranscribed. A regulatory protein binds an operator sequence in the DNA adjacent to the gene array, and controls production of the polycis-tronic (polygenic) mRNA. 5. Gene regulation in bacteria and phage is similar in many ways to the emerging information about gene regulation in eukaryotes, including humans. Much remains to be discovered; even in E. coli, one of the most closely studied organisms on earth, 35% of the genomic ORFs have no attributed function. 台大農藝系 遺傳學 601 20000

34. The lac Operon of E. coli Animation: Regulation of Expression of the lac Operon Genes 1. An inducible operon responds to an inducer substance (e.g., lactose). An inducer is a small molecule that joins with a regulatory protein to control transcription of the operon. 2. The regulatory event typically occurs at a specific DNA sequence (controlling site) near the protein-coding sequence (Figure 16.1). 3. Control of lactose metabolism in E. coli is an example of an inducible operon. 台大農藝系 遺傳學 601 20000

35. Lac Operon • Transcription is “OFF” • When there is no lactose that needs to be digested • lacI repressor is in active form  binds to operator  blocks RNA Polymerase  no transcription

36. Lac Operon • Transcription is “ON” • When there is lactose that needs to be digested • Lactose binds to lacI repressor  inactivates it • RNA Polymerase is able to bind to promoter  transcribe genes

37. Negative Regulation of Gene Expression • By default, the gene is usually switched ON. • Binding of a REPRESSOR will switch the gene OFF. • Most common regulation in BACTERIA • Often this is found as AUTOREGULATION - where too much of the gene product inhibits further transcription - usually this is through binding to the upstream promoter control region. • A good "classic" example is the E.coli lac operon. Angelia Teo Jan 09

38. Positive control of Regulation • By default, the gene is usually switched OFF. • Binding of a ACTIVATOR will switch the gene ON. (often transcriptional activators / factors bind and bend DNA upstream of the promoter.) • Most common in EUKARYOTES • Some promoters are not very functional in the absence of a transcriptional activatorprotein(s). Angelia Teo Jan 09

39. Lac Operon • Lactose metabolism occurs when the environment contains lactose. • Enzymes required for lactose degradation are TURNED ON. beta-galactosidase (lac Z) - enzyme hydrolyzes the bond between glucose & galactose. Lactose Permease (Lac Y) - enzyme spans the cell membrane - transports lactose into the cell from the outside environment. - Membrane is otherwise essentially impermeable to lactose. Thiogalactoside transacetylase (LacA) - The function of this enzyme is not known. Angelia Teo Jan 09

40. Lactose metabolism Angelia Teo Jan 09

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42. Regulatory elements in the Lac Operon Element Function Operator (LacO)binding site for repressor Promoter (LacP)binding site for RNA polymerase Repressor (LacI)codes for lac repressor protein Binds to DNA at operator and blocks binding of RNA polymerase at promoter Pipromoter for Lac I CAP binding site for cAMP/CAP complex Angelia Teo Jan 09

43. Glucose or Lactose? • A bacterium's prime source of food is glucose, since it does not have to be modified to enter the respiratory pathway. • So if both glucose & lactose are around, the bacterium will to turn off lactose metabolism in favor of glucose metabolism. • There are sites upstream of the Lac genes that respond to different glucose concentrations. Angelia Teo Jan 09

44. Presence of inducer - lactose Angelia Teo Jan 09

45. Regulation of Lac operon - depending on availability of lactose or glucose Low levels of Glucose / Catabolite repression Absence of lactose Angelia Teo Jan 09

46. Regulation of Lac Operon • When lactose is present, it acts as an inducer of the operon. Lactose enters the cell and binds to the Lac repressor, inducing a conformational change preventing the repressor from binding to the operator. This allows the RNA polymerase binding at the promoter to proceed with transcription of mRNA (LacZ, LacY & LacA) and production of enzymes for the metabolism of lactose. • When the inducer (lactose) is removed, the repressor returns to its original conformation and binds to operator, blocking the RNA polymerase from proceeding with transcription of mRNA, thus no protein is made. • The lac operon is always primed for transcription upon the addition of lactose. • When levels of glucose (a catabolite) in the cell are high, formation of cyclic AMP is inhibited. But when glucose levels drop, more cAMP forms. cAMP binds to a protein called CAP (catabolite activator protein), which is then activated to bind to the CAP binding site. This activates transcription, by increasing the binding affinity of RNA polymerase to promoter. This is called catabolite repression, a misnomer since it involves activation, but understandable since it seemed that the presence of glucose repressed all the other sugar metabolism operons. Angelia Teo Jan 09

47. The Tryptophan Operon (Positive regulation) • Trp operon contains the Tryptophan biosynthetic genes. • Trp repressor protein can bind to the operator of Trp operon • When tryptophan is high, it binds to the repressor and induces a change so that the repressor can now bind to DNA. • When tryptophan are low in the cell, tryptophan falls off the repressor, and the repressor goes back to its original conformation, losing its ability to bind to the DNA. RNA polymerase binds to the promoter and transcription proceeds, making tryptophan biosynthetic genes and replenishing the cell's supply of tryptophan. • This type of feedback inhibition of transcription is very common. ribosomal RNA can also act to repress their own synthesis. Angelia Teo Jan 09

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49. Repressible Operon: TrpOperon • Repressible Operon = Operon that is usually “ON” but can be inhibited • The Trp Operon • example of a repressible operon • Genes that code for enzymes needed to make the amino acid tryptophan

50. TrpR Gene • TrpR gene is the regulatory gene for the Trp operon • Found somewhere else on the genome • NOT part of the Trp operon • TrpR gene codes for a protein = TrpR repressor • TrpR gene is transcribed and translated separately from the Trp operon genes.