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