1. PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 15 GENE REGULATION IN EUKARYOTES

2. INTRODUCTION Eukaryotic organisms have many benefits from regulating their genes For example They can respond to changes in nutrient availability They can respond to environmental stresses 15-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

3. INTRODUCTION Gene regulation is necessary to ensure 1. Expression of genes in an accurate pattern during the various developmental stages of the life cycle Some genes are only expressed during embryonic stages, whereas others are only expressed in the adult 2. Differences among distinct cell types Nerve and muscle cells look so different because of gene regulation rather than differences in DNA content Figure 15.1 describes the steps of gene expression that are regulated in eukaryotes 15-3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

4. 15-4 Figure 15.1

5. Transcription factors are proteins that influence the ability of RNA polymerase to transcribe a given gene There are two main types General transcription factors Required for the binding of the RNA pol to the core promoter and its progression to the elongation stage Are necessary for basal transcription Regulatory transcription factors Serve to regulate the rate of transcription of nearby genes They influence the ability of RNA pol to begin transcription of a particular gene Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 15.1 REGULATORY TRANSCRIPTION FACTORS 15-5

6. Regulatory transcription factors recognize cis regulatory elements located near the core promoter These sequences are known as response elements, control elements or regulatory elements The binding of these proteins to these elements, affects the transcription of an associated gene A regulatory protein that increases the rate of transcription is termed an activator The sequence it binds is called an enhancer A regulatory protein that decreases the rate of transcription is termed a repressor The sequence it binds is called a silencer Most Eukaryotic genes are regulated by many factors This is known as combinatorial control Refer to Figure 15.2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 15-6

7. Figure 15.2 15-7 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

8. Transcription factor proteins contain regions, called domains, that have specific functions One domain could be for DNA-binding Another could provide a binding site for effector molecules A motif is a domain or portion of it that has a very similar structure in many different proteins Figure 15.3 depicts several different domain structures found in transcription factor proteins Structural Features of Regulatory Transcription Factors 15-8

9. Figure 15.3 15-9 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The recognition helix recognizes and makes contact with a base sequence along the major groove of DNA Hydrogen bonding between an  -helix and nucleotide bases is one way a transcription factor can bind to DNA

10. Figure 15.3 15-10 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Composed of one  -helix and two  -sheets held together by a zinc (Zn++) metal ion Two  -helices intertwined due to leucine motifs Alternating leucine residues in both proteins interact (“zip up”), resulting in protein dimerization Note: Helix-loop-helix motifs can also mediate protein dimerization Homodimers are formed by two identical transcription factors; Heterodimers are formed by two different transcription factors

11. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The binding of a transcription factor to an enhancer increases the rate of transcription This up-regulation can be 10- to 1,000-fold The binding of a transcription factor to a silencer decreases the rate of transcription This is called down-regulation Enhancers and Silencers 15-11

12. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Many response elements are orientation independent or bidirectional They can function in the forward or reverse orientation Most response elements are located within a few hundred nucleotides upstream of the promoter However, some are found at various other sites Several thousand nucleotides away Downstream from the promoter Even within introns! Enhancers and Silencers 15-12

13. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Most regulatory transcription factors do not bind directly to RNA polymerase Two common protein complexes that communicate the effects of regulatory transcription factors are 1. TFIID 2. Mediator Refer to Figure 15.4 TFIID and Mediator 15-13

14. Figure 15.4 15-14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A general transcription factor that binds to the TATA box Recruits RNA polymerase to the core promoter

15. Figure 15.4 15-14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Transcriptional activator stimulates the function of mediator This enables RNA pol to form a preinitiation complex It then proceeds to the elongation phase of transcription Transcriptional repressor inhibits the function of mediator Transcription is repressed STOP

16. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display There are three common ways that the function of regulatory transcription factors can be affected 1. Binding of a small effector molecule 2. Protein-protein interactions 3. Covalent modification Refer to Figure 15.5 Modulation of Regulatory Transcription Factor Functions 15-16

17. Figure 15.5 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 15-17 The transcription factor can now bind to DNA Formation of homodimers and heterodimers

18. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Regulatory transcription factors that respond to steroid hormones are termed steroid receptors The hormone actually binds to the transcription factor The ultimate action of a steroid hormone is to affect gene transcription Steroid hormones are produced by endocrine glands Secreted into the bloodstream Then taken up by cells that respond to the hormone Steroid Hormones and Regulatory Transcription Factors 15-18

19. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Cells respond to steroid hormones in different ways Glucocorticoids These influence nutrient metabolism in most cells They promote glucose utilization, fat mobilization and protein breakdown Gonadocorticoids These include estrogen and testosterone They influence the growth and function of the gonads Figure 15.6 shows the stepwise action of glucocorticoid hormones Steroid Hormones and Regulatory Transcription Factors 15-19

20. 15-20 Figure 15.6 Heat shock proteinHeat shock proteins leave when hormone binds to receptor Nuclear localization Sequence is exposed Formation of a homodimer Glucocorticoid Response Elements These function as enhancers GREs are located near dozens of different genes, so the hormone can activate many genes Transcription of target gene is activated Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

21. The CREB protein is another regulatory transcriptional factor functioning within living cells CREB is an acronym for cAMP response element-binding CREB protein becomes activated in response to cell- signaling molecules that cause an increase in cAMP Cyclic adenosine monophosphate The CREB protein recognizes a response element with the consensus sequence 5’–TGACGTCA–3’ This has been termed a cAMP response element (CRE) The CREB Protein 15-21

22. 15-22 The activity of the CREB protein Figure 15.7 Could be a hormone, neurotransmitter, growth factor, etc. Acts as a second messenger Activates protein kinase A Phosphorylated CREB binds to DNA and stimulates transcription Unphosphorylated CREB can bind to DNA, but cannot activate RNA pol

23. Changes in chromatin structure can involve changes in the structure of DNA and/or changes in chromosomal compaction These changes include 1. Gene amplification 2. Gene rearrangement 3. DNA methylation 4. Chromatin compaction Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 15.2 CHANGES IN CHROMATIN STRUCTURE 15-23 Refer to Table 15.1 Uncommon ways to regulate gene expression Common ways to regulate gene expression

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25. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The three-dimensional packing of chromatin is an important parameter affecting gene expression Chromatin is a very dynamic structure that can alternate between two conformations Closed conformation Chromatin is very tightly packed Transcription may be difficult or impossible Open conformation Chromatin is highly extended Transcription can take place Chromatin Structure 15-25

26. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Variations in the degree of chromatin packing occur in eukaryotic chromosomes during interphase During gene activation, tightly packed chromatin must be converted to an open conformation for transcription to occur Figure 15.8 shows micrographs of a chromosome from an amphibian oocyte The chromosome does not form a uniform 30 nm fiber Instead many decondensed loops radiate outward These are DNA regions whose genes are actively transcribed These chromosomes have been named lampbrush chromosomes They resemble brushes once used to clean kerosene lamps 15-26

27. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Aside from chromatin packing, a second structural issue to consider is the position of nucleosomes In chromatin, the nucleosomes are usually positioned at regular intervals along the DNA However, they have been shown to change positions in cells that normally express a particular gene But not in cells where the gene is inactive Refer to Figure 15.11 Chromatin Remodeling of the Promoter Region 15-36

28. 15-37 Changes in nucleosome position during the activation of the  -globin gene Figure 15.11 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Positioned at regular intervals from -3,000 to + 1,500 Disruption in nucleosome positioning from -500 to + 200

29. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display As discussed in Chapter 12, there are two common ways in which chromatin structure is altered 1. Covalent modification of histones 2. ATP-dependent chromatin remodeling So let’s review Figure 12.15 Chromatin Remodeling 15-38

30. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 1. Covalent modification of histones Amino terminals of histones are modified in various ways Acetylation; phosphorylation; methylation 15-39 Figure 12.15 Adds acetyl groups, thereby loosening the interaction between histones and DNA Removes acetyl groups, thereby restoring a tighter interaction

31. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 2. ATP-dependent chromatin remodeling The energy of ATP is used to alter the structure of nucleosomes and thus make the DNA more accessible 15-40 Figure 12.15 Proteins are members of the SWI/SNF family Acronyms refer to the effects on yeast when these enzyme are defective Mutants in SWI are defective in mating type switching Mutants in SNF are sucrose non-fermenters These effects may significantly alter gene expression

32. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display An important role for transcriptional activators is to recruit the aforementioned enzymes to the promoter A well-studied example of recruitment involves a gene in yeast that is involved in mating Yeast can exist in two mating types, termed a and  The gene HO encodes an enzyme that is required for the mating switch Refer to Figure 15.12 Chromatin Remodeling 15-41

33. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display DNA methylation is a change in chromatin structure that silences gene expression Carried out by the enzyme DNA methyltransferase It is common in some eukaryotic species, but not all Yeast and Drosophila have little DNA methylation Vertebrates and plants have abundant DNA methylation In mammals, ~ 2 to 7% of the DNA is methylated Refer to Figure 15.13 DNA Methylation 15-44

34. 15-45 Figure 15.13 Only one strand is methylated Both strands are methylated

35. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display DNA methylation usually inhibits the transcription of eukaryotic genes Especially when it occurs in the vicinity of the promoter In vertebrates and plants, many genes contain CpG islands near their promoters These CpG islands are 1,000 to 2,000 nucleotides long Contain high number of CpG sites In housekeeping genes The CpG islands are unmethylated Genes tend to be expressed in most cell types In tissue-specific genes The expression of these genes may be silenced by the methylation of CpG islands Methylation may change binding of transcription factors Methyl-CpG-binding proteins may recruit factors that lead to compaction of the chromatin 15-46

36. 15-47 Transcriptional silencing via methylation Figure 15.14 Transcriptional activator binds to unmethylated DNA This would inhibit the initiation of transcription Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

37. 15-48 Transcriptional silencing via methylation Figure 15.14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

38. Methylated DNA sequences are inherited during cell division May explain genomic imprinting (Chapter 7) Specific genes are methylated in gametes from mother or father Pattern of one copy of the gene being methylated and the other not is maintained in the resulting offspring Figure 15.15 illustrates a model explaining how methylation is passed from mother to daughter cell DNA Methylation is Heritable 15-49

39. Figure 15.15 An infrequent and highly regulated event DNA methylase converts hemi-methylated to fully- methylated DNA An efficient and routine event occurring in vertebrate and plant cells 15-50

40. So far, we have discussed various mechanisms that regulate the level of gene transcription In eukaryotic species, it is also common for gene expression to be regulated at the RNA level Refer to Table 15.2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 15.3 REGULATION OF RNA PROCESSING AND TRANSLATION 15-51

41. 15-52

42. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display One very important biological advantage of introns in eukaryotes is the phenomenon of alternative splicing Alternative splicing refers to the phenomenon that pre-mRNA can be spliced in more than one way In most cases, large sections of the coding regions are the same resulting in two alternative versions of a protein that have similar functions Nevertheless, there will be enough differences in amino acid sequences to provide each protein with its own unique characteristics Alternative Splicing 15-53

43. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The degree of splicing and alternative splicing varies greatly among different species Baker’s yeast contains about 6,300 genes ~ 300 (i.e., 5%) encode mRNAs that are spliced Only a few of these 300 have been shown to be alternatively spliced Humans contain ~ 25,000 genes Most of these encode mRNAs that are spliced It is estimated that about 70% are alternatively spliced Note: Certain mRNAs can be alternatively spliced to produce dozens of different mRNAs Alternative Splicing 15-54

44. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 15.16 considers an example of alternative splicing for a gene that encodes  -tropomyosin This protein functions in the regulation of cell contraction It is found in Smooth muscle cells (uterus and small intestine) Striated muscle cells (cardiac and skeletal muscle) The different cells of a multicellular organism regulate their contraction in subtly different ways One way to accomplish this is to produce different forms of  -tropomyosin by alternative splicing Alternative Splicing 15-55

45. 15-56 Figure 15.16 Alternative ways that the rat  -tropomyosin pre-mRNA can be spliced Found in the mature mRNA from all cell types Not found in all mature mRNAs These alternatively spliced versions of  -tropomyosin vary in function to meet the needs of the cell type in which they are found

46. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Alternative splicing is not a random event The specific pattern of splicing is regulated in a given cell It involves proteins known as splicing factors These play a key role in the choice of splice sites One example of splicing factors is the SR proteins At their C-terminal end, they have a domain that is rich in serine (S) and arginine (R) It is involved in protein-protein recognition At their N-terminal end, they have an RNA-binding domain Alternative Splicing 15-57

47. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The spliceosome recognizes the 5’ and 3’ splice sites and removes the intervening intron Refer to Chapter 12 Splicing factors modulate the ability of spliceosomes to recognize or choose the splice sites This can occur in two ways 1. Some splicing factors inhibit the ability of a spliceosome to recognize a splice site Refer to Figure 15.17a 2. Some splicing factors enhance the ability of a spliceosome to recognize a splice site Refer to Figure 15.17b 15-58

48. 15-59 Figure 15.17 The role of splicing factors during alternative splicing Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

49. 15-60 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 15.17 The role of splicing factors during alternative splicing

50. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The term RNA editing refers to a change in the nucleotide sequence of an RNA molecule It involves additions or deletion of particular bases Or a conversion of one type of base to another RNA editing can have various effects on mRNAs Generating start or stop codons Changing the coding sequence of a polypeptide Table 15.3 describes several examples where RNA editing has been found RNA Editing 15-61

51. 15-62

52. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display RNA editing was first discovered in trypanosomes The protozoa that cause sleeping sickness In these organisms, the process involves guide RNA Guide RNA can direct the addition or deletion of one or more uracils into an RNA Refer to Figure 15.18 RNA Editing 15-63

53. 15-64 3’ end has a sequence of uracils First, the 5’ anchor binds to target DNA Cleaves target DNA at a defined location 5’ end is complementary to mRNA being edited 3’ end of guide RNA becomes displaced from target DNA Inserts uracils Removes uracils Rejoins the two DNA pieces Figure 15.18

54. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A more widespread mechanism for RNA editing involves changes of one type of base to another This involves deamination of bases 15-65 Figure 15.19 Recognized as guanine during translation

55. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The stability of eukaryotic mRNA varies considerably Several minutes to several days The stability of mRNA can be regulated so that its half-life is shortened or lengthened This will greatly influence the mRNA concentration And consequently gene expression Factors that can affect mRNA stability include 1. Length of the polyA tail 2. Destabilizing elements Stability of mRNA 15-66

56. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 1. Length of the polyA tail Most newly made mRNA have a polyA tail that is about 200 nucleotides long It is recognized by polyA-binding protein Which binds to the polyA tail and enhances stability As an mRNA ages, its polyA tail is shortened by the action of cellular nucleases The polyA-binding protein can no longer bind if the polyA tail is less than 10 to 30 adenosines long The mRNA will then be rapidly degraded by exo- and endonucleases 15-67

57. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 2. Destabilizing elements Found especially in mRNAs that have short half-lives These elements can be found anywhere on the mRNA However, they are most common at the 3’ end between the stop codon and the polyA tail 15-68 Figure 15.20 3’-untranslated region 5’-untranslated region AU-rich element (ARE) Recognized and bound by cellular proteins These proteins influence mRNA degradation

58. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Double-stranded RNA can silence the expression of certain genes This discovery was made from research in plants and the nematode Caenorhabditis elegans Using cloning techniques, it is possible to introduce cloned genes into the genomes of plants When cloned genes were introduced in multiple copies, the expression of the gene was often silenced This may be due to the formation of double-stranded RNA. See Figure 15.21 Double-stranded RNA and Gene Silencing 15-69

59. 15-70 Gene insertion leading to the production of double-stranded RNA Figure 15.21 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display This event will silence the expression of the cloned gene

60. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Evidence for mRNA degradation via double-stranded RNA came from studies in C. elegans Injection of antisense RNA (i.e., RNA complementary to a specific mRNA) into oocytes silences gene expression Surprisingly, injection of double-stranded RNA was 10 times more potent at inhibiting the expression of the corresponding mRNA This phenomenon was termed RNA interference (RNAi) A proposed mechanism for RNAi is shown in Figure 15.22 Double-stranded RNA and Gene Silencing 15-71

61. 15-72 Figure 15.22

62. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display RNA Interference Mediated by Micro RNAs 15-74 microRNAs (miRNAs) cause RNA interference encoded by genes in eukaryotic organisms genes do not encode a protein give rise to small RNA molecules, typically 21 to 23 nucleotides Silence expression of specific mRNAs In humans, approximately 200 miRNAs have been identified A proposed mechanism for RNAi is shown in Figure 15.23

63. 15-75 Figure 15.23

64. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Presents a newly identified form of gene regulation May offer a defense mechanism against certain viruses Many RNA viruses have either double-stranded RNA genome or exist as a double-strand during their life cycle May play a role in silencing certain transposable elements Random insertion may place an element near a cellular promoter which will produce a silencing RNA Benefits of RNA interference 15-76

65. THE END

66. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Modulation of translation initiation factors is widely used to control fundamental cellular processes Under certain conditions, it is advantageous for a cell to stop synthesizing proteins Viral infection So that the virus cannot manufacture viral proteins Starvation So that the cell conserves resources Initiation Factors and the Rate of Translation 15-77

67. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The phosphorylation of initiation factors has been found to affect translation in eukaryotic cells Two initiation factors appear to play a central role in controlling the initiation of translation eIF2 and eIF4F The function of these two factors are modulated by phosphorylation in opposite ways Phosphorylation of eIF2  inhibits translation Phosphorylation of eIF4F increases the rate of translation Figure 15.24 shows the events leading to the translational inhibition by eIF2  15-78

68. 15-79 Figure 15.24 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Required if eIF2 is to promote binding of the initiator tRNA met to the 40S subunit

69. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display eIF4F provides another way to control translation It regulates the binding of mRNA to the ribosomal initiation complex eIF4F is stimulated by phosphorylation Conditions that increase its phosphorylation include signaling molecules that promote cell proliferation Growth factors and insulin, for example Conditions that decrease its phosphorylation include heat shock and viral infection 15-80

70. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Regulation of iron assimilation provides an example how the translation of specific mRNAs is modulated Iron is an essential element for the survival of living organisms It is required for the function of many different enzymes The assimilation of iron is depicted in Figure 15.25 Iron Assimilation and Translation 15-81

71. 15-82 Figure 15.25 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Protein that carries iron through the bloodstream A hollow spherical protein Prevents toxic buildup of too much iron in the cell

72. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Iron is a vital yet potentially toxic substance So mammalian cells have evolved an interesting way to regulate iron assimilation An RNA-binding protein known as the iron regulatory protein (IRP) plays a key role It influences both the ferritin mRNA and the transferrin receptor mRNA This protein binds to a regulatory element within the mRNA known as the iron response element (IRE) IRE is found in the 5’-UTR in ferritin mRNA And in the 3’-UTR in transferrin receptor mRNA Regulation of iron assimilation is shown in Figure 15.26 15-83

73. 15-84 Figure 15.26 (a) Regulation of ferritin mRNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

74. 15-85 Figure 15.26 (b) Regulation of transferrin receptor mRNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display More mRNA means more translation