6.gene expression


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6.gene expression

  1. 1. Biochemistry Chen Yonggang Zhejiang University Schools of Medicine
  2. 2. Regulation of Gene Expression: Putting information to work
  3. 3. Information encoded in DNA is of no use unless it is expressed <ul><li>Each cell has many genes but few are active at any time </li></ul><ul><li>Although all cells have the total complement of genes only a portion are expressed </li></ul><ul><li>Cells must respond to the situation at hand and do what is needed for survival </li></ul><ul><li>Cells only have so much energy and must use it efficiently </li></ul>
  4. 4. Bacteria are simpler than eucaryotic cells but have similar processes <ul><li>In trying to understand the design of living things we study bacteria as models for life </li></ul><ul><li>Once processes are understood in bacteria we find that the same processes function in eucaryotes, but with much more complexity </li></ul><ul><li>Bacteria don’t complain, need no sleep and have no protectors. In fact no one worries whether they live or die </li></ul>
  5. 5. Gene expression systems be understood by studying two models <ul><li>Biochemical processes can be subdivided into two categories </li></ul><ul><ul><li>Anabolism </li></ul></ul><ul><ul><ul><li>The process of building complexity </li></ul></ul></ul><ul><ul><li>Catabolism </li></ul></ul><ul><ul><ul><li>The process of breaking down complexity </li></ul></ul></ul><ul><li>Living things oxidize fuels and use the energy to synthesize molecules and do work </li></ul>
  6. 6. Life processes are controlled by enzymes, gene products <ul><li>For each process activated in the cell, genes must be expressed </li></ul><ul><li>The gene is transcribed to form mRNAs and proteins(enzymes) are made </li></ul><ul><li>The enzymes carry out catabolic (degradative) steps or in other cases, anabolic (synthetic) steps </li></ul>
  7. 7. The easiest bacterium to study is E. coli <ul><li>Since it lives in the human colon, there is an easy source </li></ul><ul><li>It is easy to isolate, it lives on almost any fuel source </li></ul><ul><li>It grows at a comfortable temperature (37 o C) </li></ul><ul><li>A lot is known about it including its entire chromosomal DNA sequence </li></ul>
  8. 8. The energy for growth and function comes from food <ul><li>E. coli prefers glucose as a fuel. Glucose, a sugar can be oxidized to provide the energy for ATP synthesis and to generate the Proton Motive Force, energy sources used by the bacterium to drive its life processes </li></ul><ul><li>However, in the colon it gets a variety of foods depending on the whims of its host </li></ul><ul><li>When we drink milk, E.coli needs to use lactose, milk sugar in place of glucose </li></ul>
  9. 9. E. coli does not normally express the enzymes needed to use lactose <ul><li>The genes required to utilize glucose are located together in a segment of DNA and are transcribed together to make a polycistronic mRNA, containing information for making all the enzymes needed </li></ul><ul><li>Such a coordinately expressed set of genes is called an Operon </li></ul>
  10. 10. The lac operon, an inducible operon McKee 18.28
  11. 11. The lac operon contains regulatory and structural genes <ul><li>The Operon is organized in the chromosome </li></ul><ul><li>Lac I CAP Lac P Lac O LacZ LacY LacA </li></ul><ul><li>Lac P is the Promoter for the operon </li></ul><ul><li>Lac I, CAP and LacO are regulatory genes </li></ul><ul><li>LacZ, LacY and Lac A are structural genes for the proteins needed to metabolize lactose </li></ul>
  12. 12. Lactose is a dissacharide
  13. 13. To use lactose, 3 structural genes are required at the same time <ul><li>Lac Z encodes  -galactosidase which cleaves the  -galactosidic bond joining the glucose and the galactose </li></ul><ul><li>Lac Y encodes a lactose permease, a membrane protein which transports lactose into the bacterium </li></ul><ul><li>Lac A encodes a transacetylase, an enzyme whose role is not well understood </li></ul>
  14. 14. The LacI gene encodes a control protein, a repressor <ul><li>Lac I is located upstream of the lac promoter Lac P and thus is not under regulation, it is encodes a constitutive protein, the repressor which has high affinity for the Lac O gene, called the operator </li></ul><ul><li>Normally the repressor binds to the operator forming a repressor / operator complex </li></ul>
  15. 15. The repressor/operator interferes with RNA polymerase binding <ul><li>The operator overlaps the Lac P gene so when repressor binds it blocks access of RNA polymerase to the promoter </li></ul><ul><li>The repressor blocks the transcription of the Lac operon </li></ul><ul><li>Under normal circumstances the lac operon is not expressed, no proteins are synthesized, it is a silent region of the chromosome </li></ul>
  16. 17. When lactose is available, the Lac operon is activated <ul><li>An enzyme converts a small portion of the available lactose to a modified molecule, allolactose </li></ul><ul><li>Allolactose is an inducer of the Lac operon, </li></ul><ul><li>As an inducer it turns on the operon by virture of its affinity for the repressor </li></ul><ul><li>When allolactose binds the repressor undergoes a conformational change </li></ul>
  17. 18. Induction of the lac operon McKee 18.29
  18. 19. The repressor/inducer complex does not bind to the operator <ul><li>Removal of the repressor from the operator opens the promoter for RNA polymerase binding allowing transcription of the lac operon and the utilization of lactose </li></ul><ul><li>In the laboratory, IPTG, a gratuitous inducer (is not cleaved by  -galactosidase) is used to study the process </li></ul>
  19. 20. Control of a catabolic operon Devlin 8-3
  20. 21. Mutations in genes affect operon expression <ul><li>Mutations in the Lac I gene yield a repressor which always binds the operator, such Repressor Constititive Mutations never allow Lac activation </li></ul><ul><li>Mutations in the Lac O gene yield an operator which cannot bind repressor, such Operator Constituitive Mutations never allow the Lac operon to be repressed </li></ul>
  21. 22. Glucose is a breakdown product of lactose and the preferred fuel <ul><li>E. coli prefers to use glucose </li></ul><ul><li>The presence of glucose inactivates another protein Adenyl Cyclase </li></ul><ul><li>Normally Adenyl Cyclase mediates the reaction </li></ul><ul><li>ATP 5’,3’cAMP + PPi </li></ul><ul><li>The availability of glucose reduces cAMP </li></ul><ul><li>When glucose is gone, Adenyl Cyclase is activated and cAMP is formed </li></ul>
  22. 23. CAP encodes a protein, Catabolite Activator Protein <ul><li>When lactose is the only fuel cAMP is synthesized </li></ul><ul><li>cAMP binds Catabolite Activator Protein </li></ul><ul><li>The cAMP-Catabolite Activator Protein Complex is a DNA binding protein which creates an abrupt kink in the DNA in the Lac P or promoter </li></ul><ul><li>The kink increases RNA polymerase binding to the promoter, turning on the operon </li></ul>
  23. 24. cAMP-CAP exerts positive control, enhancing transcription of the operon Devlin 8-6
  24. 25. The Lac operon is an inducible system <ul><li>Many catabolic pathways employ induction as a regulatory motif </li></ul><ul><li>An inducer, either the molecule to be catabolized or a related molecule induces the transcription of the genes needed for its catabolism </li></ul>
  25. 26. The lac operon, an inducible operon McKee 18.28
  26. 27. Anabolic, biosynthetic operons are regulated differently <ul><li>Tryptophan is an important amino acid, further the sythesis of tryptophan uses energy and critical starting materials </li></ul><ul><li>When tryptophan is available, E. coli does not need to engage in its synthesis </li></ul><ul><li>Under these conditions the Trp operon is repressed </li></ul><ul><li>Anabolic processes are repressed when not needed </li></ul>
  27. 28. The Trp operon is similar to the Lac operon in structure <ul><li>It has regulatory genes and structural genes </li></ul><ul><li>There are 5 structural genes encoding 3 enzymes needed for Tryptophan synthesis (multiple subunits for 2 enzymes) </li></ul><ul><li>The genes are arranged as seen before </li></ul><ul><li>TrpP TrpO Trpa Trp E TrpD TrpC TrpB TrpA Trpf </li></ul><ul><li>The promoter Trp P and operator Trp O are upstream from the structural genes </li></ul>
  28. 29. Trp R , not associated with the operon constituitvely synthesizes a repressor <ul><li>The trp repressor binds to the operator only when complexed with a corepressor , a small molecule(in this case, tryptophan) </li></ul><ul><li>Thus, normally the operon is turned on , however when the corepressor is available, the operator will be repressed , turned off </li></ul><ul><li>The Trp operon is a repressible operon as are many anabolic, biosynthetic operons </li></ul><ul><li>In the absence of corepressor it is derepressed </li></ul>
  29. 30. The trp operon is repressible Devlin 8-7
  30. 31. In addition to gene regulation, the pathway is regulated by enzymes <ul><li>The first enzyme in the pathway is Anthranilate Synthetase, encoded by genes TrpD and TrpE </li></ul><ul><li>As is common in anabolic pathways, this first committed enzyme in the pathway is regulated by Feedback Inhibition </li></ul><ul><li>Thus, if Tryptophan is present in the medium the enzyme is inhibited and this stops the synthesis of new tryptophan </li></ul>
  31. 32. Such enzymes, called Flux-Determining Enzymes are regulated <ul><li>Flux-determining enzymes have an active site which carries out the enzymatic function </li></ul><ul><li>They have another(allo) site(steric) which can bind the end product of the pathway </li></ul><ul><li>Binding the feedback inhibitor to the allosteric site alters the conformation of the enzyme and diminishes its activity </li></ul>
  32. 33. Tryptophan synthesis is controlled by gene expression and enzyme activity <ul><li>Ability to synthesize tryptophan is controlled by the ability to transcribe an mRNA- Repressible </li></ul><ul><li>The flux through the pathway is controlled by allosteric feedback inhibition- Inhibitable </li></ul><ul><li>The amount of tryptophan synthesized meets the needs of the bacterium </li></ul>
  33. 34. Eucaryotic Regulation <ul><li>Most eucaryotic gene expression is regulated by the same kinds of processes seen in simpler organisms </li></ul><ul><li>Induction and Repression are common model in eucaryotes </li></ul><ul><li>Because of the differences in gene organization expression does not involve operons in eucaryotes </li></ul>
  34. 35. Scientists used to believe that based on evolution, simpler is better <ul><li>As we study gene expression we find more and more complexity </li></ul><ul><li>The complexity does not appear to be careless or repetitive </li></ul><ul><li>Novel designs for precise control seem to be necessary and are almost unimaginable until discovered </li></ul><ul><li>Makes one wonder? </li></ul>
  35. 36. Eucaryotic genes are organized in logical arrays <ul><li>As in procaryotes, many eucaryotic genes are clustered by function and need </li></ul><ul><li>Ribosomal RNA genes are clustered into multiple tandemly repeated arrays </li></ul><ul><li>Histone genes are also clustered and tandemly repeated in some organisms </li></ul><ul><li>Most genes are present in single copies </li></ul>
  36. 37. Gene clustering may be related to expression <ul><li>The different globin genes, expressing the various polypeptides of hemoglobin are clustered </li></ul><ul><li>Thus during development moving from early to fetal to adult hemoglobin synthesis utilizes co-located genes </li></ul>
  37. 38. Gene expression in eucaryotes occurs from chromatin, not DNA <ul><li>During expression of genes in Drosophila segments of DNA in the polytene chromosomes become puffed during development </li></ul><ul><li>Puffing is related to the transition from condensed (heterochromatin) to dispersed chromatin(euchromatin) </li></ul><ul><li>Euchromatin is transcriptionally active </li></ul><ul><li>Transcription is hormonally induced </li></ul>
  38. 39. The structure of transcriptionally active chromatin is unique <ul><li>DNAse I digestion patterns of euchromatin and heterochromatin are different </li></ul><ul><li>When condensed, the fragments produced are larger than when dispersed </li></ul><ul><li>Covalent modification of histones, such as phosphorylation and acetylation alters digestion patterns </li></ul><ul><li>Digestion hypersensitivity is related to bound regulatory proteins </li></ul>
  39. 40. The DNA of active genes may be altered either in structure or access <ul><li>Transcriptionally active globin genes are more sensitive than quiescent genes to DNAse I digestion </li></ul><ul><li>Methylation of Cytosines is lower in actively transcribing genes. Since methylation blocks access to the major groove in DNA- demethylation could open the DNA to the binding of regulatory proteins </li></ul>
  40. 41. Gene expression is primarily controlled by transcription <ul><li>As in procaryotes, the expression of genes depends upon the transcription of information </li></ul><ul><li>Regulation of eucaryotic transcription is more complex than that for procaryotes-more components </li></ul><ul><li>Transcription requires access to DNA </li></ul><ul><li>Nucleosomes must be disrupted prior to transcription </li></ul>
  41. 42. Eucaryotic RNA polymerases cannot act alone <ul><li>Eucaryotic RNA polymerase II transcribes DNA to form hnRNA and mRNA to form active proteins </li></ul><ul><li>For RNA polymerase II to function, a preinitiation complex must be formed at the TATA box immediately upstream of the RNAP binding site </li></ul><ul><li>Formation of the preinitiation complex is dependent upon binding domains for general transcription factors (TFs) which require access to the minor groove of the DNA </li></ul>
  42. 43. DNA access can be modified <ul><li>Acetylation of histones reduces the lysine-derived positive charge on histones. Since the negative charges on the DNA backbone bind to + charged histones, DNA binding is reduced </li></ul><ul><li>Protein (SWI/SNF) complexes interact with RNA Polymerase II and ATP to open access to DNA sequences for TF binding </li></ul>
  43. 44. Methylation of cytosines blocks access to TFs Devlin 8-20
  44. 45. Activation of transcription depends on forming an initiation complex <ul><li>Activators and inhibitors of transcription alter the probability of forming the complex </li></ul><ul><li>In contrast to procaryotes where one or two proteins promote transcription at a promoter, eucaryotic regulation depends on many TFs, both general and specific factors acting at many different sites in the DNA </li></ul><ul><li>Each transcription factor has a specific role </li></ul>
  45. 46. In general, all transcription factors have two domains <ul><li>Transcription factors have a protein binding domain and a DNA binding domain and may also bind co-activators </li></ul><ul><li>DNA binding domains are sequence specific </li></ul><ul><li>DNA binding domains are conserved across species </li></ul><ul><li>Common motifs are seen throughout all eucaryotes </li></ul>
  46. 47. There are many general TFs required for transcription <ul><li>One of these factors (TFIID) is a large complex which contains among other proteins a TATA binding protein(TBP) </li></ul><ul><li>This complex serves as the foundation for the assembly of the initiation complex at the TATA box(-27 bp) </li></ul><ul><li>Binding of this complex causes a large distortion in the DNA double helix </li></ul>
  47. 48. The TATA box anchors the initation complex Devlin 8-22
  48. 49. While binding to the TATA box is essential, it is not sufficient <ul><li>Eucaryotic promoters are defined as all sequences which affect gene transcription </li></ul><ul><li>Thus eucaryotic promoters require multiple transcription factor binding sites </li></ul><ul><li>CAAT and GC boxes are often components </li></ul><ul><li>Other sequences which bind such effectors as hormone receptor binding sites are called response elements </li></ul><ul><li>Enhancers are distant factor binding sites </li></ul>
  49. 50. Eucaryotic genes can have multiple promoters <ul><li>Since “promoter” denotes all sequences involved in transcription, one gene, activated by multiple events will have multiple response elements and even enhancers </li></ul><ul><li>Thus a gene may be induced or repressed in concert with other genes in response to varying stimuli </li></ul><ul><li>Stimuli are transduced by specific transcription factors which bind to response elements and generate the induction or repression </li></ul>
  50. 51. Four common DNA binding motifs are used in TFs <ul><li>Helix-turn-helix (H-T-H) motif proteins bind in the grooves of DNA straddling the strand </li></ul><ul><li>Zinc finger motif proteins bind in the major groove of DNA and recognize specific sequences </li></ul><ul><li>Leucine zipper motif proteins form a dimer with another protein, it can scissor across and recognize DNA sequences </li></ul><ul><li>Helix-Loop-Helix (H-L-H) motif proteins are similar to HTH </li></ul>
  51. 52. Transcription factor DNA binding domains are sequence specific <ul><li>All appear to bind in the major groove </li></ul><ul><li>Most have dyad symmetry </li></ul><ul><li>All have strong secondary structure character </li></ul><ul><li>Each has variation in its expression </li></ul><ul><li>Most are very small in comparison with the transcription factor as a whole </li></ul>
  52. 53. Helix-turn-helix proteins are found in both procaryotes and eucaryotes <ul><li>The cro proteins which regulates viral lysogeny/lytic phases employs an H-T-H motif </li></ul><ul><li>All H-T-H proteins employ a 20 amino acid sequence organized into a 7 aa a helix-a 4 aa non-helical turn, followed by a 9 aa helix </li></ul><ul><li>Generally, these are repeated across a dyad axis to form a symmetrical DNA binding domain </li></ul>
  53. 54. HTH domains bind in the major groove of DNA Devlin 8-24
  54. 55. Each domain has a specific role <ul><li>The 9 amino acid helix is the DNA binding domain which recognizes the DNA sequence through interaction with hydrophobic amino acids such as valine or leucine </li></ul><ul><li>The 4 amino acid turn drapes over the polynucleotide strand </li></ul><ul><li>The 7 amino acid helix stabilizes the binding to the DNA </li></ul>
  55. 56. Specific binding occurs in the major groove Devlin 9-19,20
  56. 57. The Zinc finger motif binds specific sequences in the DNA <ul><li>Zinc fingers are so named because of the looping nature of the structure which can wrap in the groove of the DNA </li></ul><ul><li>Zinc is coordinated between 4 cysteines or 2 histidines and 2 cysteines </li></ul><ul><li>Many zinc fingers can exist within a single transcription factor, increasing the specificity of binding </li></ul>
  57. 58. Zinc fingers can wrap in the major groove of DNA Devlin 9-22
  58. 59. Zinc fingers mediate hydrogen bonding <ul><li>Sequential multiple zinc fingers can probe multiple turns of the DNA double helix, binding specific patterns of H bonds </li></ul><ul><li>Some zinc finger motifs use one finger to recognize and an adjacent finger to stabilize binding </li></ul>
  59. 60. Leucine zippers use dimeric helical molecules to bind DNA <ul><li>Leucine zipper use antiparallel proteins to scissor across the DNA double helix </li></ul><ul><li>They recognize DNA sequences and allow specific binding in the major groove </li></ul><ul><li>The leucines give rise to strong a helical structures </li></ul><ul><li>They form hydrophobic heterodimeric or homodimeric binding structures </li></ul>
  60. 61. Leucine zippers are common motifs Devlin 8-26,9-25
  61. 62. The Helix-Loop-Helix is a variation on the HTH motif <ul><li>HLH proteins such as TFIID (TATA binding) use a dyad symmetric HLH to bind specific sequences </li></ul><ul><li>Conformational changes induced by interaction with other transcription factors shift the attachment to the specific DNA sequences </li></ul>
  62. 63. HLH proteins bind to DNA sequences Devlin 8-27
  63. 64. Transcription is controlled through common mechanisms <ul><li>Primary binding of TFIID to the TATA box provides the foundation of the preinitiation complex </li></ul><ul><li>Binding of specific initiation factors to recognition sequences more remote from the gene interact through DNA folding to form the initiation complex </li></ul><ul><li>Binding of cofactors activates the transcription factors </li></ul>
  64. 65. Initiation of transcription <ul><li>Each transcription factor has one or multiple specific DNA binding domains </li></ul><ul><li>Each specific transcription factor is activated in response to a cellular event </li></ul><ul><li>The initiation complex recruits chromatin modifying enzymes such as acetylases to loosen the DNA structure </li></ul><ul><li>The complex recruits RNA polymerase II to initiate and elongate the mRNA </li></ul>
  65. 66. Transcription is precisely regulated to meet the needs of the cell Devlin 8-30
  66. 67. Questions for you to think about <ul><li>How could proto-oncogenes such as ras, fos and myc modify cell function to give rise to cancer? </li></ul><ul><li>Why are we concerned about the effects of carcinogens, UV light and retroviral infection with regard to tumor suppression? </li></ul><ul><li>What are heat shock effector elements? </li></ul><ul><li>What do Immunoglobulin heavy chain M and G genes and hemoglobin  ,  , and  chain genes have in common? (hint: different genes are expressed under different developmental and functional conditions) </li></ul>