Gene expression in prokaryotes

  • 1,118 views
Uploaded on

Regulation of Gene Expression- Types of control, responses, genes, Lac operon

Regulation of Gene Expression- Types of control, responses, genes, Lac operon

More in: Education
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Be the first to comment
    Be the first to like this
No Downloads

Views

Total Views
1,118
On Slideshare
0
From Embeds
0
Number of Embeds
1

Actions

Shares
Downloads
103
Comments
0
Likes
0

Embeds 0

No embeds

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
    No notes for slide

Transcript

  • 1. Regulation of Gene Expression-1Regulation of Gene Expression-1 (In prokaryotes)(In prokaryotes) By- Professor (Dr.) Namrata Chhabra Biochemistry For Medics- Lecture Notes www.namrata.co
  • 2. IntroductionIntroduction o Gene expression is the combined process of the transcription of a gene into mRNA, o the processing of that mRNA, and o its translation into protein (for protein- encoding genes). 03/26/14 2Biochemistry For Medics
  • 3. 03/26/14 Biochemistry For Medics 3
  • 4. Significance of gene ExpressionSignificance of gene Expression Regulated expression of genes is required for Adaptation, Differentiation and Development 03/26/14 4Biochemistry For Medics
  • 5. 1) Adaptation 1) Adaptation- Organisms adapt to environmental changes by altering gene expression. a) Bacteria are highly versatile and responsive organisms: the rate of synthesis of some proteins in bacteria may vary more than a 1000-fold in response to the supply of nutrients or to environmental challenges. 03/26/14 5Biochemistry For Medics
  • 6. 1) Adaptation b) Cells of multicellular organisms also respond to varying conditions. Such cells exposed to hormones and growth factors change substantially in shape, growth rate, and other characteristics. 03/26/14 6Biochemistry For Medics
  • 7. 2) Tissue specific differentiation and2) Tissue specific differentiation and developmentdevelopment The genetic information present in each somatic cell of a metazoan organism is practically identical. The exceptions in the genetic information are found in those few cells that have amplified or rearranged genes in order to perform specialized cellular functions. 03/26/14 7Biochemistry For Medics
  • 8. 22) Tissue specific differentiation and) Tissue specific differentiation and developmentdevelopment Cells from muscle and nerve tissue show strikingly different morphologies and other properties, yet they contain exactly the same DNA. These diverse properties are the result of differences in gene expression. Expression of the genetic information is regulated during ontogeny and differentiation of the organism and its cellular components. 03/26/14 8Biochemistry For Medics
  • 9. Control of gene ExpressionControl of gene Expression • Mammalian cells possess about 1000 times more genetic information than does the bacterium Escherichia coli. • Much of this additional genetic information is probably involved in regulation of gene expression during the differentiation of tissues • and biologic processes in the multicellular organism and in ensuring that the organism can respond to complex environmental challenges. 03/26/14 9Biochemistry For Medics
  • 10. How is gene expression controlled?How is gene expression controlled? • Gene activity is controlled first and foremost at the level of transcription. • Much of this control is achieved through the interplay between proteins that bind to specific DNA sequences and their DNA binding sites. • This can have a positive or negative effect on transcription. 03/26/14 10Biochemistry For Medics
  • 11. How is gene expression controlled?How is gene expression controlled? • Transcription control can result in tissue- specific gene expression. • In addition to transcription level controls, gene expression can also be modulated by gene amplification, gene rearrangement, posttranscriptional modifications, and RNA stabilization. 03/26/14 11Biochemistry For Medics
  • 12. Types of gene regulationTypes of gene regulation There are three types of genes regulation- • Positive • Negative and • Double negative 03/26/14 12Biochemistry For Medics
  • 13. Types of gene regulationTypes of gene regulation A) Positive regulation • When the expression of genetic information is quantitatively increased by the presence of a specific regulatory element, regulation is said to be positive. • The element or molecule mediating positive regulation is a positive regulator or activator. 03/26/14 13Biochemistry For Medics
  • 14. Types of gene regulationTypes of gene regulation B) Negative regulation • When the expression of genetic information is diminished by the presence of a specific regulatory element, regulation is said to be negative. • The element or molecule mediating negative regulation is said to be a negative regulator or repressor. 03/26/14 14Biochemistry For Medics
  • 15. Types of gene regulationTypes of gene regulation A double negative has the effect of acting as a positive. • An effector that inhibits the function of a negative regulator will bring about a positive regulation. • Many regulated systems that appear to be induced are in fact derepressed at the molecular level. 03/26/14 15Biochemistry For Medics
  • 16. Responses in Gene ExpressionResponses in Gene Expression • Type A response is characterized by an increased extent of gene expression that is dependent upon the continued presence of the inducing signal. • When the inducing signal is removed, the amount of gene expression diminishes to its basal level, • The amount repeatedly increases in response to the reappearance of the specific signal. 03/26/14 16Biochemistry For Medics
  • 17. Type A response The response is observed only in the presence of a signal 03/26/14 17Biochemistry For Medics
  • 18. Type A responseType A response • This type of response is commonly observed in prokaryotes in response to sudden changes of the intracellular concentration of a nutrient. • It is also observed in many higher organisms after exposure to inducers such as hormones, nutrients, or growth factors 03/26/14 18Biochemistry For Medics
  • 19. Type B ResponseType B Response • Type B response exhibits an increased amount of gene expression that is transient even in the continued presence of the regulatory signal. • After the regulatory signal has terminated and the cell has been allowed to recover, a second transient response to a subsequent regulatory signal may be observed. 03/26/14 19Biochemistry For Medics
  • 20. Type B ResponseType B Response The signal persists but the response is transient. 03/26/14 20Biochemistry For Medics
  • 21. Type B ResponseType B Response • This phenomenon of response- desensitization-recovery characterizes the action of many pharmacologic agents, but it is also a feature of many naturally occurring processes. • This type of response commonly occurs during development of an organism, when only the transient appearance of a specific gene product is required although the signal persists.03/26/14 21Biochemistry For Medics
  • 22. Type C ResponseType C Response • The type C response pattern exhibits, in response to the regulatory signal, • an increased extent of gene expression that persists indefinitely even after termination of the signal. • The signal acts as a trigger in this pattern. 03/26/14 22Biochemistry For Medics
  • 23. Type C ResponseType C Response • The response is signal independent. • Response persists even in the absence of a signal.03/26/14 23Biochemistry For Medics
  • 24. Type C ResponseType C Response • Once expression of the gene is initiated in the cell, it cannot be terminated even in the daughter cells; • It is therefore an irreversible and inherited alteration. • This type of response typically occurs during the development of differentiated function in a tissue or organ. 03/26/14 24Biochemistry For Medics
  • 25. Types of genes in Gene ExpressionTypes of genes in Gene Expression • Inducible gene- An inducible gene is one whose expression increases in response to an inducer or activator, a specific positive regulatory signal. • Inducible genes have relatively low basal rates of transcription. 03/26/14 25Biochemistry For Medics
  • 26. Types of genes in Gene ExpressionTypes of genes in Gene Expression • Constitutive genes-are expressed at a reasonably constant rate and are not known to be subjecedt to regulation. • These are often referred to as housekeeping genes. 03/26/14 26Biochemistry For Medics
  • 27. Types of genes in Gene ExpressionTypes of genes in Gene Expression • As a result of mutation, some inducible gene products become constitutively expressed. • A mutation resulting in constitutive expression of what was formerly a regulated gene is called a constitutive mutation. 03/26/14 27Biochemistry For Medics
  • 28. Regulation of Prokaryotic GeneRegulation of Prokaryotic Gene ExpressionExpression • Controlling gene expression is one method of regulating metabolism. • Prokaryotes must use substances and synthesize macromolecules just fast enough to meet their needs. 03/26/14 28Biochemistry For Medics
  • 29. Regulation of Prokaryotic GeneRegulation of Prokaryotic Gene ExpressionExpression • The genes for metabolizing enzymes are expressed only in the presence of nutrients. • If the enzymes are not needed, genes are turned off. • This allows for conservation of cell resources. 03/26/14 29Biochemistry For Medics
  • 30. • In prokaryotes, the genes involved in a metabolic pathway are often present in a linear array called an Operon, e.g., the lac Operon. • An Operon can be regulated by a single promoter or regulatory region. • The cistron is the smallest unit of genetic expression. Features of Prokaryotic geneFeatures of Prokaryotic gene ExpressionExpression 03/26/14 30Biochemistry For Medics
  • 31. CistronCistron • Some enzymes and other protein molecules are composed of two or more non identical subunits. • The "one gene, one enzyme" concept is not necessarily valid. • The cistron is the genetic unit coding for the structure of the subunit of a protein molecule 03/26/14 31Biochemistry For Medics
  • 32. CistronCistron • A single mRNA carries information for multiple proteins • This type of mRNA is called a polycistronic mRNA and is totally unique to prokaryotes • The polycistronic Lac Operon mRNA is translated into three separate proteins • In Eukaryotes the m-RNA is monocistronic 03/26/14 32Biochemistry For Medics
  • 33. Lac Operon ModelLac Operon Model • Jacob and Monod in 1961 described their Operon model in a classic paper. • Their hypothesis was to a large extent based on observations on the regulation of lactose metabolism by the intestinal bacterium E coli. 03/26/14 33Biochemistry For Medics
  • 34. Lac Operon – Basic conceptLac Operon – Basic concept • Bacteria such as E. coli usually rely on glucose as their source of carbon and energy. • However, when glucose is scarce, E. coli can use lactose as their carbon source even though this disaccharide does not lie on any major metabolic pathways. • An essential enzyme in the metabolism of lactose is β-galactosidase, which hydrolyzes lactose into galactose and glucose 03/26/14 34Biochemistry For Medics
  • 35. Lac Operon – Basic conceptLac Operon – Basic concept Action of Beta galactosidase on lactose, breaks lactose to galactose and glucose 03/26/14 35Biochemistry For Medics
  • 36. Inducible Lac Operon • An E. coli cell growing on a carbon source such as glucose or glycerol contains fewer than 10 molecules of β -galactosidase. • In contrast, the same cell contains several thousand molecules of the enzyme when grown on lactose. • The presence of lactose in the culture medium induces a large increase in the amount of β -galactosidase by eliciting the synthesis of new enzyme molecules rather than by activating a preexisting but inactive precursor.03/26/14 36Biochemistry For Medics
  • 37. Components of Lac OperonComponents of Lac Operon • The genetic elements of the model are a regulator gene, a regulatory DNA sequence called an operator site, and a set of structural genes. • The regulator gene encodes a repressor protein that binds to the operator site. • The binding of the repressor to the operator prevents transcription of the structural genes. • The operator and its associated structural genes constitute the Operon.03/26/14 37Biochemistry For Medics
  • 38. Components of Lac OperonComponents of Lac Operon • For the lactose (lac) Operon, the i gene encodes the repressor, o is the operator site, and the z, y, and a genes are the structural genes for β -galactosidase, the permease, and the transacetylase, respectively. • The Operon also contains a promoter site (denoted by p), which directs the RNA polymerase to the correct transcription initiation site. transcript. 03/26/14 38Biochemistry For Medics
  • 39. Components of Lac OperonComponents of Lac Operon 03/26/14 39Biochemistry For Medics
  • 40. Components of Lac OperonComponents of Lac Operon 03/26/14 40Biochemistry For Medics
  • 41. •The z, y, and a genes are transcribed to give a single mRNA molecule that encodes all three proteins. • An mRNA molecule encoding more than one protein is known as a polygenic or polycistronic Components of Lac OperonComponents of Lac Operon 03/26/14 41Biochemistry For Medics
  • 42. How does the lac repressor inhibitHow does the lac repressor inhibit the expression of the lac Operon?the expression of the lac Operon? •The lac repressor can exist as a dimer of 37-kd subunits, and two dimers often come together to form a tetramer. • In the absence of lactose, the repressor binds very tightly and rapidly to the operator. 03/26/14 42Biochemistry For Medics
  • 43. Negative Control- Repression •When the lac repressor is bound to DNA, it prevents bound RNA polymerase from locally unwinding the DNA to expose the bases that will act as the template for the synthesis of the RNA strand. •Thus, very little β-galactosidase, permease, or transacetylase are produced. 03/26/14 43Biochemistry For Medics
  • 44. Lac I Promoter gene Operator gene Lac Z Lac Y Lac A R Translation & Transcription RNA polymerase No Gene Expression 03/26/14 44Biochemistry For Medics
  • 45. How does the presence of lactose triggerHow does the presence of lactose trigger expression from theexpression from the laclac Operon?Operon? (b) Double negative control- Derepression Lactose or lactose analogue, bind to lac repressor and act as inducers of lac Operon A lactose analog that is capable of inducing the lac Operon while not itself serving as a substrate for -galactosidase is an example of a gratuitous inducer. An example is isopropylthiogalactoside (IPTG) 03/26/14 45Biochemistry For Medics
  • 46. •When the lac repressor is bound to the inducer, the repressor's affinity for operator DNA is greatly reduced. •This binding leads to local conformational changes so that it cannot easily contact DNA simultaneously, leading to a dramatic reduction in DNA-binding affinity and the release of DNA by the lac repressor. •With the operator site unoccupied, RNA polymerase can then transcribe the other lac genes and the bacterium produces the proteins necessary for the efficient utilization of lactose. (b) Double negative control- Derepression 03/26/14 46Biochemistry For Medics
  • 47. Lac I Promoter gene Operator gene Lac Z Lac Y Lac A RNA polymerase Repressor tetramer R Translation & Transcription Inactive repressor Desired product 03/26/14 47Biochemistry For Medics
  • 48. An inducer derepresses the lac Operon and allows transcription of the structural genes for β- galactosidase, galactoside permease, and thiogalactoside transacetylase.  Repressible and Inducible enzymes are both an example of negative control of a pathway.  Activating the repressor proteins shuts off the pathway.  Positive control requires that an activator molecule switch on transcription. (b) Double negative control- Derepression 03/26/14 48Biochemistry For Medics
  • 49. ( c) Positive control- CAP-cAMPc) Positive control- CAP-cAMP bindingbinding There are also DNA-binding proteins that stimulate transcription. One particularly example is the catabolite activator protein (CAP), which is also known as the cAMP response protein (CRP). Within the lac Operon, CAP binds to an inverted repeat that is centered near position -61 relative to the start site for transcription 03/26/14 49Biochemistry For Medics
  • 50. 03/26/14 Biochemistry For Medics 50
  • 51. Within the lac Operon, CAP binds to an inverted repeat that is centered near position -61 relative to the start site for transcription The CAP-cAMP complex stimulates the initiation of transcription by approximately a factor of 50. A major factor in this stimulation is the recruitment of RNA polymerase to promoters to which CAP is bound. c) Positive control- CAP-cAMP bindingc) Positive control- CAP-cAMP binding 03/26/14 51Biochemistry For Medics
  • 52. An increase in the cAMP level inside an E. coli bacterium results in the formation of CAP-cAMP complexes that bind to many promoters and stimulate the transcription of genes encoding a variety of catabolic enzymes. Thus, the CAP-cAMP regulation acts as a positive regulator because its presence is required for gene expression. c) Positive control- CAP-cAMP bindingc) Positive control- CAP-cAMP binding 03/26/14 52Biochemistry For Medics
  • 53. State of Lac Operon in the presenceState of Lac Operon in the presence of only glucoseof only glucose When grown on glucose, E. coli have a very low level of catabolic enzymes such as β- galactosidase.  It would be wasteful to synthesize these enzymes when glucose is abundant. The inhibitory effect of glucose, called catabolite repression, is due to the ability of glucose to lower the intracellular concentration of cyclic AMP. 03/26/14 53Biochemistry For Medics
  • 54. State of Lac Operon in the presence ofState of Lac Operon in the presence of only glucoseonly glucose The bacterium accumulates cAMP only when it is starved for a source of carbon.  In the presence of glucose—or of glycerol in concentrations sufficient for growth—the bacteria will lack sufficient cAMP to bind to CAP because the glucose inhibits adenylyl cyclase, the enzyme that converts ATP to cAMP. Thus, in the presence of glucose or glycerol, cAMP-saturated CAP is lacking, so that the DNA- dependent RNA polymerase cannot initiate transcription of the lac Operon. 03/26/14 54Biochemistry For Medics
  • 55. Maximum Expression of Lac OperonMaximum Expression of Lac Operon The lac Operon is controlled by two distinct DNA binding factors;  One that acts positively (cAMP-CRP complex) and The other that acts negatively (LacI repressor). Maximal activity of the lac Operon occurs when glucose levels are low (high cAMP with CAP activation) and lactose is presen,t LacI is prevented from binding to the operator).03/26/14 55Biochemistry For Medics
  • 56. Constitutive Expression andConstitutive Expression and continuous repressioncontinuous repression When the lacI gene has been mutated so that its product, LacI, is not capable of binding to operator DNA, the organism will exhibit constitutive expression of the lac Operon.  In a contrary manner, an organism with a lacI gene mutation that produces a LacI protein which prevents the binding of an inducer to the repressor will remain repressed even in the presence of the inducer molecule, because the inducer cannot bind to the repressor on the operator locus in order to derepress the Operon. Similarly, bacteria harboring mutations in their lac operator locus such that the operator sequence will not bind a normal repressor molecule constitutively express the lac Operon genes.03/26/14 56Biochemistry For Medics
  • 57. Lac I Promoter gene Operator gene Lac Z Lac Y Lac A mRNA R R RR Lactose absentRepressor molecules Repressor tetramer No Gene Expression RNA polymerase 03/26/14 57Biochemistry For Medics
  • 58. Lac I Promoter gene Operator gene Lac Z Lac Y Lac A mRNA R R RR RNA polymerase mRNA Thiogalacto side transacetyl ase Permeas e β- galactosidase Inducer Inactive repressor R Lactose/ Isopropyl Thiogalactoside (IPTG) present 03/26/14 58Biochemistry For Medics
  • 59. If there occurs no glucose metabolism 03/26/14 59Biochemistry For Medics
  • 60. Lac I Promoter gene Operator gene Lac Z Lac Y Lac A cAMP ↑↑↑ Glucose pool gets depleted due to metabolism CAP-cAMP complex formed cAMP RNA polymerase mRNA β-galactosidase Permease Thiogalactoside transacetylase If there occurs glucose metabolism R IR I 03/26/14 60Biochemistry For Medics
  • 61. Summary- Regulation of ExpressionSummary- Regulation of Expression of Lac Operonof Lac Operon 1) In the absence of lactose- Lac Operon remains repressed due to the presence of lac repressor at the operator site- (Negative control). 2) In the presence of only Lactose- Lac Operon is derepressed, the structural genes are transcribed and the lactose metabolizing enzymes are synthesized (Double negative control). 03/26/14 61Biochemistry For Medics
  • 62. Summary- Regulation of ExpressionSummary- Regulation of Expression of Lac Operonof Lac Operon 3) In the presence of both glucose and lactose- CAP -cAMP complex is not formed, RNA polymerase can not initiate the transcription of structural genes despite the fact that the operator site is vacant due to the binding of lactose/allolactose with lac repressor. Lac Operon remains in the repressed state. It is absence of positive regulation. 03/26/14 62Biochemistry For Medics
  • 63. Revision of ConceptsRevision of Concepts 1) http://bcs.whfreeman.com/thelifewire/content/chp13/1302 001.html 2) http://highered.mcgrawhill.com/sites/0072556781/student_ view0/chapter12/animation_quiz_4.html 3) http://www.youtube.com/watch? v=iPQZXMKZEfw&feature=related 03/26/14 63Biochemistry For Medics