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Gene regulation

Gene regulation. Ch 18.1-4, Campbell 9 th edition. 18.1 Gene Regulation in Prokaryotes:. Lac operon – inducible operon. Normally, the repressor IS bound to the operator, so lac operon is OFF. In the "induced" state, the lac repressor is NOT bound to the operator site .

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Gene regulation

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  1. Gene regulation Ch 18.1-4, Campbell 9th edition

  2. 18.1 Gene Regulation in Prokaryotes: Lac operon – inducible operon • Normally, the repressor IS bound to the operator, so lac operon is OFF. • In the "induced" state, the lac repressor is NOT bound to the operator site.

  3. Trp operon - repressible Trp operon is normally ON When tryptophan is present it binds to the repressor, which activates it. The activated repressor will bind to the operator, “repressing” the operon

  4. Positive Gene Regulation • Positive activation of lac operon • Bacteria uses carbohydrates for energy – glucose vs. lactose • How is this signaled to the bacteria?

  5. Positive activation is through the CAP protein, which is activated by cAMP. cAMP accumulates when glucose is scarce.

  6. 18.2 Gene Regulation in Eukaryotes • Gene is expressed when it makes a protein • Expression regulated at various levels: • Chromatin structure • Transcription factors* • Alternative splicing • Non-coding RNAs that degrade other mRNAs

  7. Chromatin packing

  8. Chromatin structure • Histone acetylation- acetyl groups are added to histones • loosens chromatin structure – promotes transcription • Deacetylation – acetyl groups removed, reduces transcription • Methylation – methyl groups added to certain bases in DNA • Reduces transcription in some species • In genomic imprinting, regulates expression of maternal or paternal alleles of certain genes

  9. Regulation of Transcription:Typical Eukaryotic Gene Organization • Control elements, segments of noncoding DNA, are associated with eukaryotic genes. Control elements act as binding sites for transcription factors that help regulate transcription • Control elements and the transcription factors that bind them allow for precise control of gene regulation

  10. Eukaryotic gene & transcript Enhancer(distal controlelements) Poly-Asignalsequence Proximalcontrolelements Transcriptionterminationregion Transcriptionstart site Exon Intron Intron Exon Exon DNA Upstream Downstream Promoter Poly-Asignal Transcription Exon Intron Intron Exon Exon Primary RNAtranscript(pre-mRNA) Cleaved3 end ofprimarytranscript 5 RNA processing Intron RNA Coding segment mRNA 3 G P P P AAA AAA Startcodon Stopcodon Poly-Atail 5 UTR 5 Cap 3 UTR

  11. Transcription factors – general ones are required for the RNA polymerase binding. They bind first to the DNA, and then recruit the RNA polymerase. Specific transcription factors bind with control elements for regulation • Enhancers – groups of control elements upstream of a gene, have binding sites for specific transcription factors

  12. Activators - a protein that binds to an enhancer and stimulates transcription of a gene • have two domains, one that binds DNA and a second that activates transcription • Repressors - transcription factors that inhibit expression of a particular gene by a variety of methods

  13. MyoD – a specific transcription factor that acts as an activator MyoD is a master regulatory gene Activationdomain DNA-bindingdomain DNA

  14. Gene Switches • http://www.hhmi.org/biointeractive/gene-switch

  15. Promoter Activators Gene DNA Distal controlelement TATA box Enhancer Generaltranscriptionfactors DNA-bendingprotein Group of mediator proteins RNApolymerase II RNApolymerase II Transcriptioninitiation complex RNA synthesis

  16. Model for Transcription Initiation • 1. Transcriptional activators bind to DNA & recruit chromatin remodeling complexes and histone acetyltransferases • 2. These open up the chromatin to expose promoter & regulatory sequences • 3. Transcriptional factors bind to enhancers • 4. DNA bending protein protein brings activators, mediator proteins, and general transcirption factors together to form transcription initiation complex on promoter

  17. Regulation of Eukaryotic DNA Transcription • http://www.hhmi.org/biointeractive/regulation-eukaryotic-dna-transcription

  18. Coordinately controlled genes in eukaryotes • Genes coding for enzymes of a metabolic pathway are often scattered over different chromosomes • Coordinate gene expression depends on simultaneous expression of the genes • Chemical signalling is often used for coordinate gene expression – i.e. hormones

  19. Alternate Gene Splicing • Post transcriptional regulation through RNA processing • Different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns

  20. Exons DNA 4 1 3 5 2 Troponin T gene PrimaryRNAtranscript 3 5 2 4 1 RNA splicing or mRNA 3 5 5 2 2 4 1 1

  21. mRNA degradation • Lifespan of mRNA in cytoplasm affects protein synthesis • mRNA in eukaryotes lasts longer than prokaryotic mRNA

  22. Translation • Initiation of translation can be blocked by proteins that bind to parts of mRNA

  23. Protein processing & degradation • Post-translational protein processing includes cleavage, and addition of functional groups • Proteasomes are giant protein complexes that bind protein molecules and degrade them

  24. 18.3 Non coding mRNAs • A large part of the genome is made up of DNA that is transcribed into non-coding mRNAs (ncRNA) • These can affect translation and chromatin expression

  25. RNA • Important in many cellular machines: • Ribosome rRNA • SpliceosomesnRNA • Telomerase telomerase RNA

  26. Interference with Translation • MicroRNAs (miRNA) are single-stranded RNA molecules that can bind to mRNA • Small Interfering RNA (siRNA) act similarly to miRNAs, but have a longer, double stranded precursor • They can degrade mRNA or block its translation • This is called RNA interference - RNAi

  27. Hairpin Hydrogenbond miRNA Dicer 5 3 (a) Primary miRNA transcript miRNA miRNA-proteincomplex mRNA degraded Translation blocked (b) Generation and function of miRNAs

  28. microRNAs • A novel class of ncRNA gene • Products are ~22 nt RNAs • Precursors are 70-100 nt hairpins • Gene regulation by pairing to mRNA • Unknown before 2001 • Forms RISC – RNA inducing silencing complex

  29. Small Interfering RNAs - siRNA • RNA interference (RNAi) – when double stranded RNA injected into the cell, it turned off expression of gene with same sequence as the RNA • siRNAs are the cause of this RNAi • Similar to miRNA, but formation is different • Many siRNAs are formed from a longer, double stranded RNA molecule • Some siRNAs can bind back to chromatin and cause changes in the chromatin

  30. siRNA

  31. Chromatin & ncRNA • In some yeasts, siRNAs can play role in heterochromatin forming, and block parts of chromosome • Small ncRNAs can induce heterochromatin, which blocks parts of chromosome, blocking transposons

  32. RNAi (~5 min)] • http://www.youtube.com/watch?v=cK-OGB1_ELE

  33. 18.4 Differential gene expression leads to different cell types in multicellular organism • One fertilized egg can give rise to many different cell types • Differential gene expression results from genes being regulated differently in each cell type • Materials in the egg can set up gene regulation that is carried out as cells divide

  34. Cell development Zygote cell – totipotent – has potential to develop into a complete organism Cell determination – cell has committed to a final fate, it is unable to change at this point Cell differentiation – cell produces tissue-specific proteins, cell has clear cut identity Morphogenesis – organization of cells into tissues & organs

  35. Cytoplasmic Determinants • Based on uneven distribution of cytoplasmic determinants in egg • The cytoplasm has RNA & proteins that were encoded by the mother’s DNA • When cell divides, the two cells have different amounts of the determinants, which can determine the cell’s fate

  36. (a) Cytoplasmic determinants in the egg Unfertilized egg Sperm Nucleus Fertilization Molecules of twodifferent cytoplasmicdeterminants Zygote(fertilized egg) Mitoticcell division Two-celledembryo

  37. Induction signals and cell differentiation Environment around the cell, especially signals from nearby embryonic cells influence development of cells The changes in gene expression lead into observable cellular changes In the process called induction, signal molecules from embryonic cells cause transcriptional changes in nearby target cells Interactions between cells induce differentiation of specialized cell types

  38. Signal Induction - types • Signals from one group of cells influence another group of cells Diffusion: signal diffuses from distance to receptor– i.e. hormone, or other signal molecule - receptor can transmit signal through second messengers in signal transduction pathway

  39. Signal Induction - types Direct contact – neighboring cells Gap junction – cytoplasm of 2 cells is connected

  40. Muscle cell determination

  41. Pattern formation • What controls the body plan of an organism? How do organs get in the right place? • 2 general models: • Morphogen gradient • Sequential induction

  42. Sequential induction • Differentiation due to production & release of a series of chemical signals

  43. Morphogen gradient A diffusible chemical signal, or morphogen, is produced. The concentration is higher closer to the source, and lower farther away from the source. The fate of the cell depends on its exposure to the different threshold levels.

  44. Drosophila- model organism • Lewis studied development by looking at mutants with bizarre developmental defects, and through this discovered homeotic genes • Homeotic genes specify the identity of body segments • Mutations in these genes lead to structures in the wrong place

  45. In a fruit fly, for example, Hox genes lay out the various main body segments—the head, thorax, and abdomen. Here we see a representation of a fruit fly embryo viewed from the side, with its anterior end to the left and with various Hox genes shown in different colors. Each Hox gene, such as the blue Ultrabithorax or Ubx gene, is expressed in different areas, or domains, along the anterior-to-posterior axis.

  46. Drosophila development • Cytoplasmic determinants in egg establish axes of drosophila body • Bicoid mRNA from mother is translated into the Bicoid protein in the Drosophila zygote • Bicoid is transcription factor that turns on genes in different levels

  47. a. Bicoid concentration & 4 genes affected b. concentration gradient of Bicoid in zygote– more at right c. concentration gradient in embryo after several divisions d. hunchback protein – green, kruppel protein - orange

  48. Eric Wieschaus – Bicoidgradient (3:28) • http://www.youtube.com/watch?v=pAoK-KOUTZM • Bicoid animation (2:15) • http://www.youtube.com/watch?v=uaedzlrnBGY

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