1. CHAPTER 12 The Cell Nucleus and the Control of Gene Expression 1

2. Cells express their genetic information selectively. Gene expression is controlled by regulatory machinery in the cell nucleus. Gene regulation -Prokaryotes -Eukaryotes ---Genomic control ---Transcriptional control ---Posttranscriptional control 2

3. 12.2 Control of Gene Expression in Bacteria Genes --Constitutive. ( i.e., organic carbon, nitrogen, phosphorus, sulfur and metal ions-iron-, Ribosomal and glycolysis genes ). ---Regulated. ( i.e., 0 2 -metabolic genes- ) ---Adaptative. ( i.e., present or absence of nutrients ) 3

4. Bacterial cells selectively express genes to use the available resources effectively. The presence of lactose in the medium indices the synthesis of the enzyme β-galactosidase. The presence of tryptophan in the medium represses the genes that encode enzymes for tryptophan synthesis. 4

5. The kinetics of β-galactosidase induction Inducible enzymes Substrate induction Catabolic Pathway 5

6. Product inhibition End-product repression Negative feedback Anabolic Pathway 6

7. Control of Gene Expression in Bacteria The Bacterial Operon – An operon is a functional complex of genes containing the information for enzymes of a metabolic pathway. It includes: Structural genes – code for the enzymes and are translated from a single mRNA ( Polycistronic). Promoter – where the RNA polymerase binds. Operator – site next to the promoter, where the regulatory protein can bind. A repressor (~proteins) which binds to a specific DNA sequence to determine whether or not a particular gene is transcribed. The regulatory gene encodes the repressor protein 7

8. Organization of a bacterial operon 8

9. Gene regulation by operons Inducible operon Allosteric protein 9

10. Control of Gene Expression in Bacteria (4) The lac Operon – It is an inducible operon, which is turned on in the presence of lactose (inducer). The lac operon contains three structural genes. Lactose binds to the repressor, changing its conformation and making it unable to bind to the operator. A repressor protein can bind to the operator and prevent transcription in the absence of lactose. 10

11. The cAMP Receptor Protein (CRP) and Its Function [Glucose] [cAMP] Glucose indirectly inhibits the adenyl cyclase 11

12. Repressible operon The Tryptophan (trp) Operon of E. coli 12

13. Control of Gene Expression in Bacteria (6) The trp Operon – It is a repressible operon, which is turned off in the presence of tryptophan. – The trp operon repressor is active only when it is bound to a corepressor such as tryptophan. 13

14. Control of Gene Expression in Bacteria (7) Riboswitches – A number of bacterial mRNAs can bind to a small metabolite (molecules), which in turn alters the gene involved in the production of such metabolite. – These mRNAs are called riboswitches because they undergo a conformational change and can suppress gene expression. – Riboswitches allow bacteria to regulate gene expression in response to some metabolites. 14

15. Riboswitches= small molecules regulating gene expression by interacting mRNA Prokaryotes and plants 15

16. Control of Gene Expression in Eukaryotes (2) Genes are turned on and off as a result of interaction with regulatory proteins. – Each cell type contains a unique set of proteins. – Regulation of gene expression occurs on three levels: Transcriptional-level control Processing-level control Translational-level control 16

17. -Eukaryotes ---Genomic control ---Transcriptional control Transcriptional-level control Processing-level control ---Posttranscriptional control 17

18. -Eukaryotes ---Genomic control ----- gene amplification (i.e., in same type of cancers--EGFR, IGFR) -----gene deletion (i.e., red cells no DNA) -----gene rearrangements (i.e., IgG) -----Chromatin (de)condensation -----DNA metylation -----Histone acetylation ---Transcriptional control ---Posttranscriptional control 18

19. The cell nucleus 19

20. 12.1 The Nucleus of a Eukaryotic Cell (1) The contents of the nucleus are enclosed by the nuclear envelope. A typical non-dividing nucleus includes: – Chromosomes as extended fibers of chromatin. – Nucleoli for rRNA synthesis. – Nucleoplasm as the fluid where solutes are dissolved. – The nuclear matrix, which is the protein- containing fibrillar network. 20

21. The Nucleus of a Eukaryotic Cell (2) The Nuclear Envelope – The nuclear envelope is a structure that divides the nucleus from its cytoplasm. It consists of two membranes separated by a nuclear space. The two membranes are fuses at sites forming a nuclear pore. The inner surface of the nuclear envelope is lined by the nuclear lamina. 21

22. The nuclear envelope 22

23. The Nucleus of a Eukaryotic Cell (3) The nuclear lamina – Support the nuclear envelope. – It is composed of lamins. – The integrity of the nuclear lamina is regulated by phosphorylation and dephosphorylation. 23

24. The nuclear lamina Hutchinson–Gilford progeria syndrome: One point mutant in Lamin A/C, abnormal growth and aging 24

25. The Nucleus of a Eukaryotic Cell (4) The Structure of the Nuclear Pore Complex and its Role in Nucleocytoplasmic Exchange – Proteins and RNA are transported in and out of the nucleus. – Nuclear pores contain the nuclear pore complex (NPC) that appears to fill the pore like a stopper. – NPC is composed of ~30 proteins called nucleoporins. 25

26. Movement of materials though the nuclear pore 26

27. A model of the vertebrate NPC Amino acids 27

28. The Nucleus of a Eukaryotic Cell (5) Proteins synthesized in the cytoplasm are targeted for the nucleus by the nuclear localization signal (NLS). – Proteins with an NLS stretch bind to an NLS receptor (importin). – Conformation of the NPC changes as the protein passes through. – RNAs move through the NPCs as RNPs and carry NES (nuclear export signals) to pass through. 28

29. Importing proteins from the cytoplasm into the nucleus NLS NES 29

30. The Nucleus of a Eukaryotic Cell (6) Chromosomes and Chromatin – Packaging the Genome Chromosomes consist of chromatin fibers, composed of DNA and associated proteins. Each chromosome contains a single, continuous linear DNA molecule. 30

31. The Nucleus of a Eukaryotic Cell (8) DNA and histones are organized into repeating subunits called nucleosomes. Each nucleosome includes a core particle of supercoiled DNA containing H2A, H2B, H3, and H4. Histone H1 functions as a linker. The histone core complex consists of two molecules each of H2A, H2B, H3, and H4 forming an octamer. – The protein component of chromosomes include histones, a group of highly conserved proteins. – Histones have a high content of basic amino acids. 31

32. Nucleosomal organization of chromatin 32

33. Levels of organization of chromatin 33

34. The Nucleus of a Eukaryotic Cell (11) Heterochromatin and Euchromatin – Euchromatin: active DNA – Heterochromatin: inactive DNA Constitutive heterochromatin remains condensed all the time (centromeres and telomeres). Consists of highly repeated sequences and few active genes (DNA stability). Facultative heterochromatin is inactivated during certain phases of the organism’s life. X chromosomes inactivation during mammalian embryogenesis.embryogenesis. 34

35. Sensitivity of Active Genes in Chromatin to Digestion with DNase I -----Chromatin decondensation Decondensation ~ gene transcription Hypersensitive sites (DNAase) 35

36. The Nucleus of a Eukaryotic Cell (9) Histone and DNA modification is one mechanism to alter the character of nucleosomes. DNA and histones are held together by noncovalent bonds. 36

37. The Nucleus of a Eukaryotic Cell (13) – The histone code hypothesis states that the activity of a chromatin region depends on the degree of chemical modification of histone tails. – Histone tail modifications influence chromatin in two ways: Serve as docking sites to recruit nonhistone proteins. Alter the way in which histones of neighboring nucleosomes interact with one another. 37

38. -----DNA metylation Methyl groups can be added to the DNA Specificity: Cytosines 5’-CG-3’ Enzyme: methyl transferase Function: gene inactivation Epigenetic changes (i.e., function)----Epigenomic 38

39. -----Histone acetylation Acetyl groups can be added to the Histones Specificity: H3 and H4 Enzyme: Acetyl transferase Function: gene activation The prefect combination for gene inactivation DNA methylation H3/H4 de-acetylation and metylation (heterochromatin) Heterochromatin protein (HP)1 39

40. HDAC: histone De-acetylase complex HAT: histone acetyltrasnferase http://www.youtube.com/watch?v=4b-NSWm24BA 40

41. The Nucleus of a Eukaryotic Cell (16) Telomeres – The end of each chromosome is called a telomere and is distinguished by a set of repeated sequences. – New repeats are added by a telomerase, a reverse transcriptase that synthesizes DNA from a RNA template. – Telomeres are required for the complete replication of the chromosome because they protect the ends from being degraded. – Telomerase activity is thought to have major effects on cell life 41

42. Telomeres 42

43. The role of telomerase 43

44. The Nucleus of a Eukaryotic Cell (22) The Nuclear Matrix – The nuclear matrix is a network of protein- containing fibrils. – It serves as more than a skeleton to maintain the shape of the nucleus and anchoring the machinery involved in nuclear activities. 44

45. Control of Gene Expression in Eukaryotes (2) Genes are turned on and off as a result of interaction with regulatory proteins. – Each cell type contains a unique set of proteins. – Regulation of gene expression occurs on three levels: -Eukaryotes ---Genomic control ---Transcriptional control Transcriptional-level control Processing-level control ---Posttranscriptional control 45

46. ---Transcriptional control -----differential gene transcription -----control elements (transcriptional factors) 46

47. 12.4 Transcriptional-level control (1) 1. Differential transcription is the most important mechanism by which eukaryotic cells determine which proteins are synthesized. 47

48. Using a DNA Microarray to Profile the Patterns of Gene Expression in Two Different Cell Types 48

49. Transcriptional-level control (3) The Role of Transcription Factors in Regulating Gene Expression – Transcription factors are the proteins that either acts as transcription activators or transcription inhibitors. A single gene can be controlled by different regulatory proteins. A single DNA-binding protein may control the expression of many different genes. 49

50. Transcriptional-level control (5) The Structure of Transcription Factors – Transcription factors contain a DNA-binding domain and an activation domain. 50

51. Transcriptional-level control (6) 1 2 3 4 51

52. Anatomy of a Typical Eukaryotic Gene, with Its Core Promoter and Proximal Control Region -----control elements Enhancer Silencer monocistronic 52

53. Model for Enhancer Action DHAC: De-acetylase complex MTC: Methyl transferase complex Co-repressor HAT: histone acetyl transferase Co-activator Remodeling complex Suppressor or silencer 53

54. Combinatorial Model for Gene Expression 54

55. -Eukaryotes ---Genomic control ---Transcriptional control ---Post-transcriptional control ----alternative splicing ----translational ----degradation of mRNA ----protein degradation 55

56. Figure 26.2 Eukaryotic genes often contain intervening sequencings (introns) that separate the exons and must be removed for proper protein translation to occur. Removal of introns involves a complicated “ molecular device ” called a spliceosome that involves protein and RNA (like a ribosome). Splicing allows an increase in the information content of a genome due to alternative splicing products that produce novel proteins. Typical mammalian gene has 7-8 exons over ~16 kb Exons are short (~100-200 bp) Introns are long (~>1kb) Pre-mRNA mRNA Splicing occurs in the nucleus 56

57. Nuclear Splice Junctions Are Short Sequences Splice sites are the sequences immediately surrounding the exon–intron boundaries. They are named for their positions relative to the intron. 57

58. The 5′ splice site at the 5′ (left) end of the intron includes the consensus sequence GU. The 3′ splice site at the 3′ (right) end of the intron includes the consensus sequence AG. The GU-AG rule describes the requirement for these constant dinucleotides at the first two and last two positions of introns in pre-mRNAs. Figure 26.3 Splice sites are generic. GT…AG (DNA)-(~98 %);(~2% ?) Directional (one direction) The apparatus for splicing is not tissue specific (with some exceptions). The mechanism by which pairs of splice sites are correctly identified is unknown. 58

59. 12.7 Post-translational Control: Determining Protein Stability (1) The factors that control a protein’s lifetime are not well understood. Protein stability may be determined by the amino acids on the N-terminus. Degradation of proteins is carried out within hollow, cylindrical proteasomes. 59

60. Post-translational Control: Determining Protein Stability (2) Proteasomes recognize proteins linked to ubiquitin ( small protein) Ubquitin is transferred by ubiquitin ligases to proteins being degraded. Once polubiquitanated, a protein is recognized by the cap of the proteasome. Once degraded, the component amino acids are released back into the cytosol. 60

61. Proteasome structure 61

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