1. Regulation of Gene Expression Chapter 18

4. Gene expression  Flow of genetic information  Genotype to phenotype  Genes to proteins  Proteins not made at random  Specific purposes  Appropriate times

5. Control of gene expression  Selective expression of genes  All genes are not expressed at the same time  Expressed at different times

6. Prokaryote regulation

7. Control of gene expression  Regulate at transcription  Gene expression responds to  Environmental conditions  Type of nutrients  Amounts of nutrients  Rapid turn over of proteins

8. Fig. 18-2 Regulation of gene expression trpE gene trpD gene trpC gene trpB gene trpA gene (b) Regulation of enzyme production (a) Regulation of enzyme activity Enzyme 1 Enzyme 2 Enzyme 3 Tryptophan Precursor Feedback inhibition

9. Prokaryote  Anabolism:  Building up of a substance  Catabolism:  Breaking apart a substance

10. Prokaryote  Operon  Section of DNA  Enzyme-coding genes  Promoter  Operator  Sequence of nucleotides  Overlaps promoter site  Controls RNA polymerase access to the promoter

12. Figure 18.3a Promoter DNA trpR Regulatory gene RNA polymerase mRNA 5′5′ 3′3′ Protein Inactive repressor mRNA 5′ (a) Tryptophan absent, repressor inactive, operon on Promoter trp operon Genes of operon trpE trpDtrpCtrpBtrpA Operator Start codon Stop codon Polypeptide subunits that make up enzymes for tryptophan synthesis EDCB A

13. Prokaryote  Multiple genes are expressed in a single gene expression  trp operon – Trytophan – Synthesis  Lac operon – Lactose – Degradation

14. Prokaryote  trp Operon:  Control system to make tryptophan  Several genes that make tryptophan  Regulatory region

15. Fig. 18-3a Polypeptide subunits that make up enzymes for tryptophan synthesis mRNA 5 RNA polymerase Promoter trp operon Genes of operon Operator Stop codon Start codon mRNA trpA 5 trpE trpD trpCtrpB AB CD E

16. Prokaryote  ⇧ tryptophan present  Bacteria will not make tryptophan  Genes are not transcribed  Enzymes will not be made  Repression

17. Prokaryote  Repressors  Proteins  Bind regulatory sites (operator)  Prevent RNA polymerase attaching to promoter  Prevent or decrease the initiation of transcription

18. Prokaryote  Repressors  Allosteric proteins  Changes shape  Active or inactive

19. Prokaryote  ⇧ tryptophan  Tryptophan binds the trp repressor  Repressor changes shape  Active shape  Repressor fits DNA better  Stops transcription  Tryptophan is a corepressor

20. Fig. 18-3b-2 (b) Tryptophan present, repressor active, operon off Tryptophan (corepressor) No RNA made Active repressor mRNA Protein DNA

22. Prokaryote  ⇩ tryptophan  Nothing binds the repressor  Inactive shape  RNA polymerase can transcribe

23. Fig. 18-3a Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on DNA mRNA 5 ProteinInactive repressor RNA polymerase Regulatory gene Promoter trp operon Genes of operon Operator Stop codon Start codon mRNA trpA 5 3 trpRtrpE trpD trpCtrpB AB CD E

25. Prokaryote  Lactose  Sugar used for energy  Enzymes needed to break it down  Lactose present  Enzymes are synthesized  Induced

26. Prokaryote  lac Operon  Promoter  Operator  Genes to code for enzymes  Metabolize (break down) lactose

27. Prokaryote  Lactose is present  Repressor released  Genes expressed  Lactose absent  Repressor binds DNA  Stops transcription

29. Prokaryote  Allolactose:  Binds repressor  Repressor releases from DNA  Inducer  Transcription begins  Lactose levels fall  Allolactose released from repressor  Repressor binds DNA blocks transcription

30. Fig. 18-4b (b) Lactose present, repressor inactive, operon on mRNA Protein DNA mRNA 5 Inactive repressor Allolactose (inducer) 5 3 RNA polymerase Permease Transacetylase lac operon  -Galactosidase lacY lacZlacAlac I

31. Fig. 18-4a (a) Lactose absent, repressor active, operon off DNA Protein Active repressor RNA polymerase Regulatory gene Promoter Operator mRNA 5 3 No RNA made lac I lacZ

34. Prokaryote  Lactose & tryptophan metabolism  Adjustment by bacteria  Regulates protein synthesis  Response to environment  Negative control of genes  Operons turned off by active repressors  Tryptophan repressible operon  Lactose inducible operon

35. Prokaryote

36.  Activators:  Bind DNA  Stimulate transcription  Involved in glucose metabolism  lac operon

37. Prokaryote  Activator:  Catabolite activator protein (CAP)  Stimulates transcription of operons  Code for enzymes to metabolize sugars  cAMP helps CAP  cAMP binds CAP to activate it  CAP binds to DNA (lac Operon)

38. Prokaryote  Glucose elevated cAMP low  cAMP not available to bind CAP  Does not stimulate transcription  Bacteria use glucose  Preferred sugar over others.

40. Prokaryote  lac operon  Regulated by positive & negative control  Low lactose  Repressor blocks transcription  High lactose  Allolactose binds repressor  Transcription happens

41. Prokaryote  lac operon  Glucose also present  CAP unable to bind  Transcription will proceed slowly  Glucose absent  CAP binds promoter  Transcription goes quickly

43. Figure 18.5 Promoter DNA Operator Promoter DNA CAP-binding site cAMP Active CAP Inactive CAP RNA polymerase binds and transcribes lac I I Allolactose Inactive lac repressor (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized lacZ CAP-binding site RNA polymerase less likely to bind Operator Inactive CAP Inactive lac repressor (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized

44. Eukaryote gene expression  All cells in an organism have the same genes  Some genes turned on  Others remain off  Leads to development of specialized cells  Cellular differentiation

45. Eukaryote gene expression  Gene expression assists in regulating development  Homeostasis  Changes in gene expression in one cell helps entire organism

46. Control of gene expression  Chromosome structure  Transcriptional control  Posttranscriptional control

47. Fig. 18-6 DNA Signal Gene NUCLEUS Chromatin modification Chromatin Gene available for transcription Exon Intron Tail RNA Cap RNA processing Primary transcript mRNA in nucleus Transport to cytoplasm mRNA in cytoplasm Translatio n CYTOPLASM Degradation of mRNA Protein processing Polypeptide Active protein Cellular function Transport to cellular destination Degradation of protein Transcription

48. Eukaryotes  1. DNA is organized into chromatin  2. Transcription occurs in nucleus  3. Each gene has its own promoter

50. Chromatin structure  DNA is tightly packaged  Heterochromatin:  Tightly packed  Euchromatin:  Less tightly packed  Influences gene expression  Promoter location  Modification of histones

51. Chromatin structure  Histone acetylation  Acetyl groups (-COCH 3 )  Attach to Lysines in histone tails  Loosen packing  Histone methylation  Methyl groups (-CH 3 )  Tightens packing

52. Fig. 18-7 Histone tails DNA double helix (a) Histone tails protrude outward from a nucleosome Acetylated histones Amino acids available for chemical modification (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription Unacetylated histones

53. Chromatin structure  Methylation of bases (cytosine)  Represses transcription  Embryo development

54. Eukaryotes  Epigenetic change:  Chromatin modifications  Change in gene expression  Passed on to the next generation  Not a DNA sequence change

55. Transcription control  RNA polymerase must bind DNA  Proteins regulate by binding DNA  RNA polymerase able to bind or not  Stimulates transcription or blocks it

56. Fig. 18-8-3 Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA Exon Intron Exon Intron Cleaved 3 end of primary transcript Primary RNA transcript Poly-A signal Transcription 5 RNA processing Intron RNA Coding segment mRNA 5 Cap 5 UTR Start codon Stop codon 3 UTR Poly-A tail 3

57. Eukaryotes  Transcription  RNA Polymerase  Transcription factors (regulatory proteins)  General transcription factors (initiation complex)  Specific transcription factors

58. Eukaryotes  Initiation of transcription  Activator proteins  Activator binds the enhancers  Enhancers (DNA sequences)  Interacts with the transcription factors  Binds to the promoter  RNA polymerase binds and transcription begins

59. Fig. 18-9-2 Enhancer TATA box Promoter Activators DNA Gene Distal control element Group of mediator proteins DNA-bending protein General transcription factors

60. Fig. 18-9-3 Enhancer TATA box Promoter Activators DNA Gene Distal control element Group of mediator proteins DNA-bending protein General transcription factors RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis

62. Eukaryotes

63. Fig. 18-10 Control elements Enhancer Available activators Albumin gene (b) Lens cell Crystallin gene expressed Available activators LENS CELL NUCLEUS LIVER CELL NUCLEUS Crystallin gene Promoter (a) Liver cell Crystallin gene not expressed Albumin gene expressed Albumin gene not expressed

64. Post transcriptional control  RNA processing  Primary transcript:  Exact copy of the entire gene  RNA splicing  Introns removed from the mRNA  snRNP’s (small nuclear ribonulceoproteins)

67. Post transcriptional control  Splicing plays a role in gene expression  Exons can be spliced together in different ways.  Leads to different polypeptides  Originated from same gene

68. Post transcriptional control  Example in humans  Calcitonin & CGRP  Hormones released from different organs  Derived from the same transcript

69. Fig. 18-11 or RNA splicing mRNA Primary RNA transcript Troponin T gene Exons DNA

70. Post transcriptional control

71.  Transport of transcript  Passes through nuclear pores  Active transport  Cannot pass until all splicing is done

72. Post transcriptional control  mRNA degradation  Life span  Some can last hours, a few weeks  mRNA for hemoglobin survive awhile

73. Post transcriptional control

74.  Translation of RNA  Translation factors are necessary  Regulate translation  Translation repressor proteins  Stop translation  Bind transcript  Prevents it from binding to the ribosome

75. Post transcriptional control

76.  Ferritin (iron storage)  Aconitase:  Translation repressor protein  Binds ferritin mRNA  Iron will bind to aconitase  Aconitase releases the mRNA  Ferritin production increases Post transcriptional control

77.  Protein modification  Phosphorylation  Other alterations can affect the activity of protein  Insulin  Starts out as a larger molecule  Cut into more active sections

78. Post transcriptional control  Protein modification  Degradation  Protein is marked by small protein  Protein complex then breaks down proteins  Proteasomes

79. Post transcriptional control

81. Fig. 18-UN4 Genes in highly compacted chromatin are generally not transcribed. Chromatin modification DNA methylation generally reduces transcription. Histone acetylation seems to loosen chromatin structure, enhancing transcription. Chromatin modification Transcription RNA processing Translation mRNA degradation Protein processing and degradation mRNA degradation Each mRNA has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs. Protein processing and degradation by proteasomes are subject to regulation. Protein processing and degradation Initiation of translation can be controlled via regulation of initiation factors. Translation ormRNA Primary RNA transcript Alternative RNA splicing: RNA processing Coordinate regulation: Enhancer for liver-specific genes Enhancer for lens-specific genes Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription. Transcription Regulation of transcription initiation: DNA control elements bind specific transcription factors.

82. Post transcriptional control  Most gene regulation-transcription  New discovery  Small RNA’s  21-28 nucleotides long  Play a role in gene expression  New transcript before leaving the nucleus

83.  RNA interference  RNA forming double stranded loops from newly formed mRNA  Loops are formed  Halves have complementary sequences  Loops inhibit expression of genes  Where double RNA came from Post transcriptional control

84.  Dicer:  Cuts double stranded RNA into smaller RNA’s called  microRNA (miRNA)  Small interfering RNA (siRNA’s)

85. Fig. 18-13 miRNA- protein complex (a) Primary miRNA transcript Translation blocked Hydrogen bond (b) Generation and function of miRNAs Hairpin miRNA Dicer 3 mRNA degraded 5

86. Post transcriptional control  miRNA’s bind mRNA  Prevents translation  siRNA’s breaks apart mRNA before it’s translated

87. Post transcriptional control  siRNAs play a role in heterochromatin formation  Block large regions of the chromosome  Small RNAs may also block transcription of specific genes

88. Fig. 18-UN5 Chromatin modification RNA processing Translation mRNA degradation Protein processing and degradation mRNA degradation miRNA or siRNA can target specific mRNAs for destruction. miRNA or siRNA can block the translation of specific mRNAs. Transcription Small RNAs can promote the formation of heterochromatin in certain regions, blocking transcription. Chromatin modification Translation

89. Embryonic development  Zygote gives rise to many different cell types  Cells → tissues →  organs → organ systems  Gene expression  Orchestrates developmental programs of animals

90. Fig. 18-14a (a) Fertilized eggs of a frog

93. Embryonic development  Zygote to adult results  Cell division  Cell differentiation:  Cells become specialized in structure & function  Morphogenesis:  “creation of from”  Body arrangement

94. Fig. 47-6 (a) Fertilized egg(b) Four-cell stage(c) Early blastula(d) Later blastula

95. Fig. 47-1 1 mm

96. Fig. 46-17 (a) 5 weeks (b) 14 weeks (c) 20 weeks

97. Embryonic development  All cells same genome  Differential gene expression  Genes regulated differently in each cell type

98. Fig. 18-10 Control elements Enhancer Available activators Albumin gene (b) Lens cell Crystallin gene expressed Available activators LENS CELL NUCLEUS LIVER CELL NUCLEUS Crystallin gene Promoter (a) Liver cell Crystallin gene not expressed Albumin gene expressed Albumin gene not expressed

99. Embryonic development  Specific activators  Materials in egg cytoplasm  Not homogeneous  Set up gene regulation  Carried out as cells divide

100. Embryonic development  Cytoplasmic determinants  Maternal substances in the egg  Influence early development  Zygote divides by mitosis  Cells contain different cytoplasmic determinants  Leads to different gene expression

101. Fig. 18-15a (a) Cytoplasmic determinants in the egg Two different cytoplasmic determinants Unfertilized egg cell Sperm Fertilization Zygote Mitotic cell division Two-celled embryo Nucleus

102. Embryonic development  Environment around cell influences development  Induction:  Signals from nearby embryonic cells  Cause transcriptional changes in target cells  Interactions between cells induce differentiation of specialized cell types

103. Fig. 18-15b (b) Induction by nearby cells Signal molecule (inducer) Signal transduction pathway Early embryo (32 cells) NUCLEUS Signal receptor

105. Embryonic development  Determination:  Observable differentiation of a cell  Commits a cell to its final fate  Cell differentiation is marked by the production of tissue-specific proteins  Gives cell characteristic structure & function

106. Embryonic development  Myoblasts:  Produce muscle-specific proteins  Form skeletal muscle cells  MyoD  One of several “master regulatory genes”  Produces proteins  Commit cells to becoming skeletal muscle

107. Embryonic development  MyoD protein  Transcription factor  Binds to enhancers of various target genes  Causes expression

108. Fig. 18-16-1 Embryonic precursor cell Nucleus OFF DNA Master regulatory gene myoD Other muscle-specific genes OFF

109. Fig. 18-16-2 Embryonic precursor cell Nucleus OFF DNA Master regulatory gene myoD Other muscle-specific genes OFF mRNA MyoD protein (transcription factor) Myoblast (determined)

110. Fig. 18-16-3 Embryonic precursor cell Nucleus OFF DNA Master regulatory gene myoD Other muscle-specific genes OFF mRNA MyoD protein (transcription factor) Myoblast (determined) mRNA Myosin, other muscle proteins, and cell cycle– blocking proteins Part of a muscle fiber (fully differentiated cell) MyoD Another transcription factor

111. Embryonic development  Pattern formation:  Development of spatial organization of tissues & organs  Begins with establishment of the major axes  Positional information:  Molecular cues control pattern formation  Tells a cell its location relative to the body axes & neighboring cells

112. Figure 18.24 G protein Growth factor Receptor Protein kinases Transcription factor (activator) NUCLEUS Protein that stimulates the cell cycle Transcription factor (activator) NUCLEUS Overexpression of protein Ras MUTATION GTP Ras protein active with or without growth factor. PP PP PP 1 3 2 5 4 6

113. Figure 18.25 Protein kinases DNA damage in genome Active form of p53 Transcription DNA damage in genome UV light Defective or missing transcription factor. Inhibitory protein absent Protein that inhibits the cell cycle NUCLEUS MUTATION 1 34 2 5

114. Fruit fly  Unfertilized egg contains cytoplasmic determinants  Determines the axes before fertilization  After fertilization,  Embryo develops into a segmented larva with three larval stages

115. Fig. 18-17a Thorax HeadAbdomen 0.5 mm Dorsal Ventral Right Posterior Left Anterior BODY AXES (a) Adult

116. Fig. 18-17b Follicle cell Nucleus Egg cell Nurse cell Egg cell developing within ovarian follicle Unfertilized egg Fertilized egg Depleted nurse cells Egg shell Fertilization Laying of egg Body segments Embryonic development Hatching 0.1 mm Segmented embryo Larval stage (b) Development from egg to larva 1 2 3 4 5

117. Fruit fly  Homeotic genes:  Control pattern formation in late embryo,larva and adult

118. Fig. 18-18 Antenna Mutant Wild type Eye Leg

119. Fruit fly  Maternal effect genes:  Encode for cytoplasmic determinants  Initially establish the axes of the body of Drosophila  Egg-polarity genes:  Maternal effect genes  Control orientation of the egg  Consequently the fly

120. Fruit Fly  Bicoid gene  Maternal effect gene  Affects the front half of the body  An embryo whose mother has a mutant bicoid gene  Lacks the front half of its body  Duplicate posterior structures at both ends

121. Fig. 18-19a T1T2 T3 A1 A2 A3A4 A5 A6 A7 A8 A7 A6 A7 Tail Head Wild-type larva Mutant larva (bicoid) EXPERIMENT A8