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  2. Control of Gene Expression in Eukaryotes Eukaryotic gene expression is usually controlled at the level of initiation of transcription. the promoter enhancers the TATA box silencers GC box Methylation CCAAT box (called the CAT box) Hormonal Control

  3. Thinking about Gene Regulation • Humans begin life from a single cell; all the genetic information needed to create an adult is in our genome. • Embryonic cells undergo differentiation to produce specific cell types such as muscle, nerve, and blood cells. • Different cell types are the consequence of differential gene expression.

  4. A typical differentiated mammalian cell makes about 10,000 proteins from approximately 30,000 genes. • Most of these are housekeeping proteins needed to maintain all cell types. • Certain proteins can only be detected in specific cell types. • How is gene expression regulated?

  5. COORDINATELY CONTROLLED GENES • PROCARYOTES • Clustered in operons • Same regulatory sites • Single mRNA molecule • Translated together • EUCARYOTES • NO operons ! • Genes individually transcribed • Own promoter • Specific enhancers for each gene

  6. (B) In procaryotes the production of mRNA molecules is much simpler. The 5' end of an mRNA molecule is produced by the initiation of transcription by RNA polymerase, and the 3' end is produced by the termination of transcription. Since procaryotic cells lack a nucleus, transcription and translation take place in a common compartment. In fact, translation of a bacterial mRNA often begins before its synthesis has been completed.

  7. Genes have regulatory DNA sequencesupstream from the initiation site where transcription begins. • The promoter is the RNA polymerase binding site. • Gene regulatory proteins bind to regulatory DNA sequences and can either prevent or enhance RNA polymerase binding. Activators in enhancer Activators in distal promoter Co-activators

  8. Independent domains bind DNA and activate transcription • DNA-binding activity and transcription-activation are carried by independent domains of an activator. • The role of the DNA-binding domain is to bring the transcription-activation domain into the vicinity of the promoter.

  9. Acidic activators: They have multiple negative charges Yeast activators: GAL4 , GCN4 Herpes Simplex Virus: VP16

  10. The modular structure of a gene activator protein. Outline of an experiment that reveals the presence of independent DNA-binding and transcription-activating domains in the yeast gene activator protein Gal4. (A) The normal activation of gene transcription produced by the Gal4 protein. (B) The chimeric gene regulatory protein requires the LexA protein DNA-binding site for its activity.

  11. Identification of Protein-Protein Interactions by the Yeast 2-Hybrid System

  12. The two hybrid techniques tests the ability of two proteins to interact by incorporating then into hybrid proteins where one has a DNA-binding domain and the other has a transcription-activating domain.

  13. ON Gene X promoter Yeast Two Hybrid Assay Yeast Gal4 • Transcription factor • Zinc Finger DNA binding domain (DBD) • Transcriptional Activation Domain (AD) AD DBD

  14. Bait AD Prey DBD The prey: from random cDNA’s which encodes for various proteins. Do any interact with the bait? The bait: cDNA of the protein you want to find partners for. OFF OFF Reporter Gene Reporter Gene promoter promoter ON Reporter Gene Yeast cells turn blue promoter Yeast Two Hybrid Assay

  15. Yeast Two Hybrid Assay Uetz, 2001

  16. Activators interact with the basal apparatus • DNA-binding domain determines the specificity of activators for the target promoter or enhancer. • DNA-binding domain is responsible for localizing a transcription-activating domain in the proximity of the basal appapratus. • An activator that works directly has a DNA-binding domain and an activating domain. • An activator does not have an activating domain may work by binding a coactivator that has an activating domain. • Several factors in the basal apparatus are targets with which activators or coactivators interact.

  17. An activator may bind a co-activator that contacts the basal apparatus.

  18. Activators may work at different stages of initiation, by contacting the TAFs of TFIID or contacting TFIIB.

  19. How does an activator stimulate transcription? Two models: The recruitment model: Activators sole effect is to increase the concentration of RNA polymerases to the promoter. Alternative model: Activator induces some change in the transcriptional complex, i.e. conformational change in enzyme, increases its efficiency.

  20. RNA polymerase may be associated with various alternative sets of transcription factors in the form of a holoenzyme complex.

  21. A model for the action of some eukaryotic transcriptional activators. The gene activator protein, bound to DNA in the rough vicinity of the promoter, facilitates the assembly of some of the general transcription factors. Although some activator proteins may be dedicated to particular steps in the pathway for transcription initiation many seem to be capable of acting at several steps.

  22. Some Promoter Binding Proteins are Repressors • Repression is usually achieved by affecting chromatin structure, but there are repressors that act by binding to specific promoters and function like trans-acting (such as bacterial repressors) to block transcription. • The global repressor NC2/Dr1/DRAP1, is a heterodimer that binds to TBP to prevent it from interacting with other components of the basal apparatus.

  23. In embryo A transcription complex involves recognition of several elements in the sea urchin H2B promoter in testis. Binding of the CAAT displacement factor (CDP) in embryo prevents the CAAT-binding factor from binding, so an active complex cannot form.

  24. Take away message: The function of a protein in binding to a known promoter element cannot be assumed: It may be activator, a repressor, or even irrelevant to gene transcription.

  25. Transcription factors are activated in several ways The activators are classified according to their The activity of an inducible activator may be regulated by several ways

  26. Oncogenes that code for transcription factors have mutations that inactivate transcription (v-erbA and possibly v-rel) or that activate transcription (v-jun and v-fos).

  27. Gene Regulatory Proteins Bind to DNA Expression depends on; cell type its environment its age extracellular signals Transcription is controlled by proteins binding to regulatory DNA sequences. Promotor includes RNA polymerase binding site initiation site Regulatory DNA sequences bound by gene regulatory proteins some short – simple gene switches some long and complex (eukaryotic) molecular microprocessors which respond to a variety of signals, integrate them, and determine the rate of transcription. Edge of bases

  28. Gene Regulatory Proteins Bind to DNA Gene regulatory proteins insert into the major groove, contacts and binds (non-covalently) to the edges of the bases (about 20 interaction), usually without disrupting the hydrogen bonds. This binding is very strong and very specific for the nucleotide sequence. Frequently DNA-binding proteins contain alpha-helices bind in pairs - dimerization - which doubles the contact area, increasing the strength and specificity of the interaction.

  29. There are many types of DNA binding domains • The activators are classified according to their DNA binding domain. • Zinc Finger • Steroid Receptors • Helix-turn-helix motif • Homeodomain • Helix-loop-helix • Leucine zippers • Members of the same group have sequence variations of a specific motif that confer specificity for individual target sites.

  30. DNA binding zinc-finger motifs • One or more zinc atoms are added to the structure. • It is found in in a cluster with additional zinc fingers, arranged one after another, the a helix of each contact the major groove of the DNA, foring a continous strech of a helices along the groove. • Two a helices are packed together with zinc atoms. Mostly found in the large family of intracellular receptor proteins (dimers). • Cys2/His2 Finger Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His

  31. Transcription factor SP1 has a series of three zinc fingers each with a characteristic pattern of cysteine and histidine residues that constitute the zinc-binding site.

  32. ~23 aa 7-8 aa Zinc fingers may form a-helices that insert into the major groove, associated with b-sheets on the other site. Cys2/His2 finger

  33. DNA binding by a zinc finger motif: The zinc-finger motif is composed of an alpha helix and a beta sheet. Each zinc-finger is held together by a molecule of zinc.

  34. A dimer of the zinc finger domain of the intracellular receptor family bound bound to its specific DNA sequence. (e.g., glucocorticoid receptor)

  35. b-sheet can also recognized DNA: i.e. bacterial met repressor. It is a dimeric protein. Each dimers b sheet can bind to the major goorve of the DNA.

  36. DNA binding by a zinc finger protein: This protein recognizes DNA using three zinc fingers of the cys-cys-his-his type arranged as direct repeats.

  37. Sequence specific interactions between different six fingers and their DNA recognition sequences.

  38. Steroid receptors have several independent domains All the receptors have independent domains for DNA binding and hormone binding.

  39. Steroid receptors have zinc fingers The first finger of a steroid receptor controls which DNA sequence is bound (red); The second finger controls spacing between the sequences (blue).