2. AP Biology 2007-2008 Control of Eukaryotic Genes

3. AP Biology The BIG Questions… How are genes turned on & off in eukaryotes? How do cells with the same genes differentiate to perform completely different, specialized functions?

4. AP Biology Evolution of gene regulation Prokaryotes  single-celled  evolved to grow & divide rapidly  must respond quickly to changes in external environment exploit transient resources Gene regulation  turn genes on & off rapidly flexibility & reversibility  adjust levels of enzymes for synthesis & digestion

5. AP Biology Evolution of gene regulation Eukaryotes  multicellular  evolved to maintain constant internal conditions while facing changing external conditions homeostasis  regulate body as a whole growth & development  long term processes specialization  turn on & off large number of genes must coordinate the body as a whole rather than serve the needs of individual cells

6. AP Biology Points of control The control of gene expression can occur at any step in the pathway from gene to functional protein 1.packing/unpacking DNA 2.transcription 3.mRNA processing 4.mRNA transport 5.translation 6.protein processing 7.protein degradation

7. AP Biology How do you fit all that DNA into nucleus?  DNA coiling & folding double helix nucleosomes chromatin fiber looped domains chromosome from DNA double helix to condensed chromosome 1. DNA packing

8. AP Biology Nucleosomes “Beads on a string”  1 st level of DNA packing  histone proteins 8 protein molecules positively charged amino acids bind tightly to negatively charged DNA DNA packing movie 8 histone molecules

9. AP Biology DNA packing as gene control Degree of packing of DNA regulates transcription  tightly wrapped around histones no transcription genes turned off  heterochromatin darker DNA (H) = tightly packed  euchromatin lighter DNA (E) = loosely packed H E

10. AP Biology DNA methylation Methylation of DNA blocks transcription factors  no transcription  genes turned off  attachment of methyl groups (–CH 3 ) to cytosine C = cytosine  nearly permanent inactivation of genes ex. inactivated mammalian X chromosome = Barr body

11. AP Biology Histone acetylation Acetylation of histones unwinds DNA  loosely wrapped around histones enables transcription genes turned on  attachment of acetyl groups (–COCH 3 ) to histones conformational change in histone proteins transcription factors have easier access to genes

12. AP Biology 2. Transcription initiation Control regions on DNA  promoter nearby control sequence on DNA binding of RNA polymerase & transcription factors “base” rate of transcription  enhancer distant control sequences on DNA binding of activator proteins “enhanced” rate (high level) of transcription

13. AP Biology Model for Enhancer action Enhancer DNA sequences  distant control sequences Activator proteins  bind to enhancer sequence & stimulates transcription Silencer proteins  bind to enhancer sequence & block gene transcription Turning on Gene movie

14. AP Biology Transcription complex Enhancer Activator Coactivator RNA polymerase II A B F E H TFIID Core promoter and initiation complex Activator Proteins regulatory proteins bind to DNA at distant enhancer sites increase the rate of transcription Coding region T A Enhancer Sites regulatory sites on DNA distant from gene Initiation Complex at Promoter Site binding site of RNA polymerase

15. AP Biology 3. Post-transcriptional control Alternative RNA splicing  variable processing of exons creates a family of proteins

16. AP Biology 4. Regulation of mRNA degradation Life span of mRNA determines amount of protein synthesis  mRNA can last from hours to weeks RNA processing movie

17. AP Biology RNA interference Small interfering RNAs (siRNA)  short segments of RNA (21-28 bases) bind to mRNA create sections of double-stranded mRNA “death” tag for mRNA  triggers degradation of mRNA  cause gene “silencing” post-transcriptional control turns off gene = no protein produced NEW! siRNA

18. AP Biology Action of siRNA siRNA double-stranded miRNA + siRNA mRNA degraded functionally turns gene off Hot…Hot new topic in biology mRNA for translation breakdown enzyme (RISC) dicer enzyme

19. AP Biology siRNA clip https://www.youtube.com/watch?v=Fa4sk YBJHoI

20. AP Biology 5. Control of translation Block initiation of translation stage  regulatory proteins attach to 5' end of mRNA prevent attachment of ribosomal subunits & initiator tRNA block translation of mRNA to protein Control of translation movie

21. AP Biology 6-7. Protein processing & degradation Protein processing  folding, cleaving, adding sugar groups, targeting for transport Protein degradation  ubiquitin tagging  proteasome degradation Protein processing movie

22. AP Biology Ubiquitin “Death tag”  mark unwanted proteins with a label  76 amino acid polypeptide, ubiquitin  labeled proteins are broken down rapidly in "waste disposers" proteasomes 1980s | 2004 Aaron Ciechanover Israel Avram Hershko Israel Irwin Rose UC Riverside

23. AP Biology Proteasome Protein-degrading “machine”  cell’s waste disposer  breaks down any proteins into 7-9 amino acid fragments cellular recycling play Nobel animation

24. AP Biology CENTRAL DOGMA Genetic information always goes from DNA to RNA to protein Gene regulation has been well studied in E. coli When a bacterial cell encounters a potential food source it will manufacture the enzymes necessary to metabolize that food

25. AP Biology CENTRAL DOGMA Genetic information always goes from DNA to RNA to protein Gene regulation has been well studied in E. coli When a bacterial cell encounters a potential food source it will manufacture the enzymes necessary to metabolize that food

26. AP Biology Gene Regulation In addition to sugars like glucose and lactose E. coli cells also require amino acids One essential aa is tryptophan. When E. coli is swimming in tryptophan (milk & poultry) it will absorb the amino acids from the media When tryptophan is not present in the media then the cell must manufacture its’ own amino acids

27. AP Biology Trp Operon E. coli uses several proteins encoded by a cluster of 5 genes to manufacture the amino acid tryptophan All 5 genes are transcribed together as a unit called an operon, which produces a single long piece of mRNA for all the genes RNA polymerase binds to a promoter located at the beginning of the first gene and proceeds down the DNA transcribing the genes in sequence

28. AP Biology Fig. 16.6

29. AP Biology GENE REGULATION In addition to amino acids, E. coli cells also metabolize sugars in their environment In 1959 Jacques Monod and Fracois Jacob looked at the ability of E. coli cells to digest the sugar lactose

30. AP Biology GENE REGULATION In the presence of the sugar lactose, E. coli makes an enzyme called beta galactosidase Beta galactosidase breaks down the sugar lactose so the E. coli can digest it for food It is the LAC Z gene in E coli that codes for the enzyme beta galactosidase

31. AP Biology Lac Z Gene The tryptophan gene is turned on when there is no tryptophan in the media That is when the cell wants to make its’ own tryptophan E. coli cells can not make the sugar lactose They can only have lactose when it is present in their environment Then they turn on genes to beak down lactose

32. AP Biology GENE REGULATION The E. coli bacteria only needs beta galactosidase if there is lactose in the environment to digest There is no point in making the enzyme if there is no lactose sugar to break down It is the combination of the promoter and the DNA that regulate when a gene will be transcribed

33. AP Biology initiation of transcription 1 mRNA splicing 2 mRNA protection 3 initiation of translation 6 mRNA processing 5 1 & 2. transcription - DNA packing - transcription factors 3 & 4. post-transcription - mRNA processing - splicing - 5’ cap & poly-A tail - breakdown by siRNA 5. translation - block start of translation 6 & 7. post-translation - protein processing - protein degradation 7 protein processing & degradation 4 4 Gene Regulation

34. AP Biology GENE REGULATION This combination of a promoter and a gene is called an OPERON Operon is a cluster of genes encoding related enzymes that are regulated together

35. AP Biology GENE REGULATION Operon consists of  A promoter site where RNA polyerase binds and begins transcribing the message  A region that makes a repressor Repressor sits on the DNA at a spot between the promoter and the gene to be transcribed This site is called the operator

36. AP Biology

37. LAC Z GENE EE.coli regulate the production of Beta Galactocidase by using a regulatory protein called a repressor The repressor binds to the lac Z gene at a site between the promotor and the start of the coding sequence The site the repressor binds to is called the operator.

38. AP Biology

39. LAC Z GENE Normally the repressor sits on the operator repressing transcription of the lac Z gene In the presence of lactose the repressor binds to the sugar and this allows the polymerase to move down the lac Z gene

40. AP Biology LAC Z GENE This results in the production of beta galactosidase which breaks down the sugar When there is no sugar left the repressor will return to its spot on the chromosome and stop the transcription of the lac Z gene

41. AP Biology

43. Concept 18.5: Cancer results from genetic changes that affect cell cycle control The gene regulation systems that go wrong during cancer are the very same systems involved in embryonic development © 2011 Pearson Education, Inc.

44. AP Biology Types of Genes Associated with Cancer Cancer can be caused by mutations to genes that regulate cell growth and division Tumor viruses can cause cancer in animals including humans © 2011 Pearson Education, Inc.

45. AP Biology Oncogenes are cancer-causing genes Proto-oncogenes are the corresponding normal cellular genes that are responsible for normal cell growth and division Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle © 2011 Pearson Education, Inc.

46. AP Biology Figure 18.23 Proto-oncogene DNA Translocation or transposition: gene moved to new locus, under new controls Gene amplification: multiple copies of the gene New promoter Normal growth- stimulating protein in excess Point mutation: within a control element within the gene Oncogene Normal growth- stimulating protein in excess Hyperactive or degradation- resistant protein

47. AP Biology Proto-oncogenes can be converted to oncogenes by  Movement of DNA within the genome: if it ends up near an active promoter, transcription may increase  Amplification of a proto-oncogene: increases the number of copies of the gene  Point mutations in the proto-oncogene or its control elements: cause an increase in gene expression © 2011 Pearson Education, Inc.

48. AP Biology Tumor-Suppressor Genes Tumor-suppressor genes help prevent uncontrolled cell growth Mutations that decrease protein products of tumor-suppressor genes may contribute to cancer onset Tumor-suppressor proteins  Repair damaged DNA  Control cell adhesion  Inhibit the cell cycle in the cell-signaling pathway © 2011 Pearson Education, Inc.

49. AP Biology Interference with Normal Cell- Signaling Pathways Mutations in the ras proto-oncogene and p53 tumor-suppressor gene are common in human cancers Mutations in the ras gene can lead to production of a hyperactive Ras protein and increased cell division © 2011 Pearson Education, Inc.

50. AP Biology Figure 18.24 Growth factor 1234512 Receptor G protein Protein kinases (phosphorylation cascade) NUCLEUS Transcription factor (activator) DNA Gene expression Protein that stimulates the cell cycle Hyperactive Ras protein (product of oncogene) issues signals on its own. (a) Cell cycle–stimulating pathway MUTATION Ras GTP P P P P P P (b) Cell cycle–inhibiting pathway Protein kinases UV light DNA damage in genome Active form of p53 DNA Protein that inhibits the cell cycle Defective or missing transcription factor, such as p53, cannot activate transcription. MUTATION EFFECTS OF MUTATIONS (c) Effects of mutations Protein overexpressed Cell cycle overstimulated Increased cell division Protein absent Cell cycle not inhibited 3

51. AP Biology Growth factor 1 Receptor G protein Protein kinases (phosphorylation cascade) NUCLEUS Transcription factor (activator) DNA Gene expression Protein that stimulates the cell cycle Hyperactive Ras protein (product of oncogene) issues signals on its own. (a) Cell cycle–stimulating pathway MUTATION Ras GTP P P P P P P 2345 Ras GTP Figure 18.24a

52. AP Biology Figure 18.24b (b) Cell cycle–inhibiting pathway Protein kinases UV light DNA damage in genome Active form of p53 DNA Protein that inhibits the cell cycle Defective or missing transcription factor, such as p53, cannot activate transcription. MUTATION 213

53. AP Biology Suppression of the cell cycle can be important in the case of damage to a cell’s DNA; p53 prevents a cell from passing on mutations due to DNA damage Mutations in the p53 gene prevent suppression of the cell cycle © 2011 Pearson Education, Inc.

54. AP Biology Figure 18.24c EFFECTS OF MUTATIONS (c) Effects of mutations Protein overexpressed Cell cycle overstimulated Increased cell division Protein absent Cell cycle not inhibited

55. AP Biology The Multistep Model of Cancer Development Multiple mutations are generally needed for full-fledged cancer; thus the incidence increases with age At the DNA level, a cancerous cell is usually characterized by at least one active oncogene and the mutation of several tumor-suppressor genes © 2011 Pearson Education, Inc.

56. AP Biology Figure 18.25 Colon Normal colon epithelial cells Loss of tumor- suppressor gene APC (or other) 1 2 345 Colon wall Small benign growth (polyp) Activation of ras oncogene Loss of tumor- suppressor gene DCC Loss of tumor- suppressor gene p53 Additional mutations Malignant tumor (carcinoma) Larger benign growth (adenoma)

57. AP Biology Figure 18.25a Colon Normal colon epithelial cells Colon wall

58. AP Biology Figure 18.25b Small benign growth (polyp) Loss of tumor- suppressor gene APC (or other) 1

59. AP Biology Figure 18.25c Activation of ras oncogene Loss of tumor-suppressor gene DCC Larger benign growth (adenoma) 2 3

60. AP Biology Figure 18.25d Loss of tumor-suppressor gene p53 Additional mutations Malignant tumor (carcinoma) 4 5

61. AP Biology Inherited Predisposition and Other Factors Contributing to Cancer Individuals can inherit oncogenes or mutant alleles of tumor-suppressor genes Inherited mutations in the tumor-suppressor gene adenomatous polyposis coli are common in individuals with colorectal cancer Mutations in the BRCA1 or BRCA2 gene are found in at least half of inherited breast cancers, and tests using DNA sequencing can detect these mutations © 2011 Pearson Education, Inc.

62. AP Biology Figure 18.26

63. AP Biology Figure 18.UN01 Operon Promoter Genes RNA polymerase Operator Polypeptides A BC A B C

64. AP Biology Figure 18.UN02 Genes expressed Genes not expressed Promoter Genes Operator Corepressor Inactive repressor: no corepressor present Active repressor: corepressor bound

65. AP Biology Figure 18.UN03 Genes expressed Genes not expressed Promoter Genes Operator Active repressor: no inducer present Inactive repressor: inducer bound

66. AP Biology Figure 18.UN04 Chromatin modification Genes in highly compacted chromatin are generally not transcribed. Histone acetylation seems to loosen chromatin structure, enhancing transcription. DNA methylation generally reduces transcription. mRNA degradation Each mRNA has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs. Regulation of transcription initiation: DNA control elements in enhancers bind specific transcription factors. Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription. Coordinate regulation: Enhancer for liver-specific genes Enhancer for lens-specific genes Transcription RNA processing Alternative RNA splicing: Primary RNA transcript mRNAor Initiation of translation can be controlled via regulation of initiation factors. Protein processing and degradation by proteasomes are subject to regulation. Translation Protein processing and degradation Chromatin modification Transcription RNA processing mRNA degradation Translation Protein processing and degradation

67. AP Biology Chromatin modification Genes in highly compacted chromatin are generally not transcribed. Histone acetylation seems to loosen chromatin structure, enhancing transcription. DNA methylation generally reduces transcription. Regulation of transcription initiation: DNA control elements in enhancers bind specific transcription factors. Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription. Coordinate regulation: Enhancer for liver-specific genes Enhancer for lens-specific genes Transcription RNA processing Alternative RNA splicing: Primary RNA transcript mRNAor Chromatin modification Transcription RNA processing mRNA degradation Translation Protein processing and degradation Figure 18.UN04a

68. AP Biology mRNA degradation Each mRNA has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs. Initiation of translation can be controlled via regulation of initiation factors. Protein processing and degradation by proteasomes are subject to regulation. Translation Protein processing and degradation Chromatin modification Transcription RNA processing mRNA degradation Translation Protein processing and degradation Figure 18.UN04b

69. AP Biology Figure 18.UN05 Chromatin modification Transcription RNA processing mRNA degradation Translation Protein processing and degradation Chromatin modification Translation mRNA degradation miRNA or siRNA can target specific mRNAs for destruction. miRNA or siRNA can block the translation of specific mRNAs. Small or large noncoding RNAs can promote the formation of heterochromatin in certain regions, blocking transcription.

70. AP Biology Figure 18.UN06 Enhancer Promoter Gene 1 Gene 2 Gene 3 Gene 4 Gene 5

71. AP Biology Figure 18.UN07 Enhancer Promoter Gene 1 Gene 2 Gene 3 Gene 4 Gene 5

72. AP Biology Figure 18.UN08 Enhancer Promoter Gene 1 Gene 2 Gene 3 Gene 4 Gene 5