1. Gene Regulation Turning genes ON and OFF

2. All cells in an organism contain the same DNA, so why are the cells different?

3. BECAUSE, they contain and are made up of different proteins.

4. BECAUSE, different genes are transcribed in the different cells so different mRNA is transcribed for protein synthesis.

5. Genes are turned on and off. When a gene is on, it is expressed meaning it is transcribed. The DNA must unzip in order for transcription—it just doesn’t have to unzip entirely!

6. All cells have “housekeeping” genes. These genes code for proteins necessary for metabolism, structure, and synthesis.

7. Some genes are tissue-specific and are only expressed in certain types of cells or tissues.

8. HOW do genes turn ON and OFF?

9. To conserve ENERGY, it is most efficient to control this at the transcription level. Why transcribe mRNA that is not needed by the cell?

10. Transcription factors: These bind to the DNA. Activators & Repressors. This grey line will represent DNA throughout this lesson.

11. Genes that are regulated by the same repressor or activator have the same or closely related regulatory sequences.

12. DNA regulatory sequences are tissue-specific. Muscle-specific genes all have a particular sequence in their DNA to which a muscle-specific transcription factor binds and activates expression of these genes. Nerve-specific genes have a DIFFERENT sequence in the DNA and different transcription factors. These DNA base sequences lie “upstream” from the base sequences that will be transcribed when the gene is expressed.

13. More about those DNA sequences

14. DNA-Binding domains have been conserved throughout evolution Transcription factors are proteins They have distinct regions with specific functions One region is the DNA-binding domain—it IS a region of the protein that is the transcription factor

15. Often the domain is an α-helix This red thing, that looks like a stick of dynamite, is just another representation of the α-helix.

16. These are the 2 most common DNA- binding domains. If you can name them & explain their function in an essay, it’s often worth a point!

17. The binding domains of repressors and activators contact specific nucleotide sequences in DNA.

18. The regulatory region of a gene has many binding sites upstream from the promoter. Often these sites are near the promoter, BUT they can be distant as well. The nucleotide sequence to which a positive activator binds is called an activator site. The nucleotide sequence to which a negative repressor binds is called a repressor site.

20. Repressors prevent transcription by being a “roadblock” so RNA polymerase cannot bind to the DNA at the promoter.

21. When a repressor is NOT bound, RNA polymerase can bind to the promoter and transcription begins.

22. Activators help RNA polymerase bind to the DNA. Very active genes have several RNA polymerases transcribing them.

23. The structure and function of the transcription factor can be altered when other molecules bind to them.

24. INDUCERS Small molecules, INDUCERS, can bind to a transcription factor [repressor in this picture], cause a conformational shape change and decrease its ability to bind DNA. This causes the repressor to abandon the DNA and transcription is no longer blocked!

25. KNOW THIS WELL!

26. OPERONS An OPERON is a group of genes that are transcribed together. They are turned ON or OFF as a unit. These 3 genes are needed to metabolize lactose. The repressor site of an operon is called an operator Lactose  glucose + galactose These genes code for the enzymes that metabolize the above reaction! These genes are only turned ON if lactose is present. Why waste energy synthesizing proteins that serve NO function in the absence of lactose?

27. Here the lac repressor is bound to the repressor site, called the operator [since it controls the operon]. It is the “roadblock” that blocks the initiation of transcription at the promoter.

28. Allolactose is an INDUCER When lactose is present, some of it exists as an isomer called allolactose. Allolactose acts as an inducer. It binds to the Lac Repressor, causing a conformational shape change.

29. This is called negative regulation of the lac operon The change in shape, causes the Lac Repressor to release the DNA so transcription is no longer blocked. RNA polymerase can now bind to the promoter and transcription of the lactose operon can begin.

30. Once more, with feeling! IN THE ABSENCE OF LACTOSE, it is normal for the lac repressor to be bound to the operator. When lactose is present, it’s isomer— allolactose—acts as an inducer. It binds to the repressor, changing its shape. This causes the repressor to release the DNA RNA polymerase can now bind to the promoter so transcription of the 3 genes comprising the operon can commence! Synthesis of the 3 proteins necessary for the breakdown of lactose are now manufactured and lactose can be utilized by the cell. This process is called NEGATIVE REGULATION OF E. coli LACTOSE METABOLISM

31. ACTIVATORS An activator steps up the transcription rate More of the lactose enzymes can be transcribed at once— assembly line style! This process is POSITIVE Regulation of the Lac Operon!

32. Catabolite Activator Protein: CAP CAP is the name of the catabolite activator protein It needs cyclic AMP [cAMP; ATP  ADP  AMP] bound to it before it can have the proper shape in order to bind to the activator site of the DNA. Once bound, it INCREASES the ability of RNA polymerase to bind to the promoter and transcribe the genes

33. This is hyper drive!

34. The cAMP-CAP complex is very attractive to RNA polymerase! Notice that the cAMP- CAP complex contacts BOTH the DNA at the activator site AND the RNA polymerase. This contact makes the RNA polymerase have MORE attraction for the DNA, stepping up the rate of transcription of the operon.

35. What is the purpose? With this regulation, cells are very E efficient! These cells would rather use glucose as a food source. That’s why it is normal for the repressor to be in place. As long as glucose is available, the cell won’t metabolize lactose. Glucose yields more energy to the cells than lactose. Glucose helps the cells grow! As it is used up, the number of dividing cells stabilizes in the population.

36. Glucose availability regulates cAMP production. These are bacterial cells—glycolysis is their only source of ATP. When the glucose is gone, ATP production ceases. ATP  ADP  AMP SO, the concentration of the cyclic form of AMP will increase

37. What if both glucose and lactose are present? Cells will use glucose first until gone! cAMP production increases as a result cAMP binds to the CAP and the activator complex, in turn, causes RNA polymerase to crank up the rate of transcription of the lac operon The enzymes necessary to utilize lactose as an energy source are QUICKLY manufactured! Explain this!

38. Negative control is caused by a repressor, which can be affected by an inducer. Positive control is caused by and activator. Repressors, inducers and activators are transcription factors.

39. Which transcription factors are bound to the DNA when the different sugars are present?

40. No lactose? The repressor is bound.

41. Lactose present? The allolactose inducer causes the bound repressor to change shape and release the DNA. The “roadblock” is removed. Transcription begins.

42. Only lactose present? The absence of glucose causes the cAMP level to rise and allows the binding of the activator [cAMP- CAP complex]. RNA polymerase’s affinity for DNA is enhanced.

43. Gene expression in eukaryotes has two main differences from the same process in prokaryotes. First, the typical multicellular eukaryotic genome is much larger than that of a bacterium. Second, cell specialization limits the expression of many genes to specific cells. HOW IS THIS DIFFERENT IN EUKARYOTES? Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

44. The estimated 35,000 genes in the human genome includes an enormous amount of DNA that does not program the synthesis of RNA or protein. This DNA is elaborately organized. –Not only is the DNA associated with protein to form chromatin, but the chromatin is organized into higher organizational levels. Level of packing is one way that gene expression is regulated. –Densely packed areas are inactivated. –Loosely packed areas are being actively transcribed. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

45. While the single circular chromosome of bacteria is coiled and looped in a complex, but orderly manner, eukaryotic chromatin is far more complex. Eukaryotic DNA is precisely combined with large amounts of protein. Chromatin structure is based on successive levels of DNA packing Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

46. During interphase of the cell cycle, chromatin fibers are usually highly extended within the nucleus. During mitosis, the chromatin coils and condenses to form short, thick chromosomes. Chromatin structure is based on successive levels of DNA packing

47. Eukaryotic chromosomes contain an enormous amount of DNA relative to their condensed length. –Each human chromosome averages about 2 x 10 8 nucleotide pairs. –If extended, each DNA molecule would be about 6 cm long, thousands of times longer than the cell diameter. –This chromosome and 45 other human chromosomes fit into the nucleus. –This occurs through an elaborate, multilevel system of DNA packing. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

48. Histone proteins are responsible for the first level of DNA packaging. –Their positively charged amino acids bind tightly to negatively charged DNA. –The five types of histones are very similar from one eukaryote to another and are even present in bacteria. Unfolded chromatin has the appearance of beads on a string, a nucleosome, in which DNA winds around a core of histone proteins. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

50. The beaded string seems to remain essentially intact throughout the cell cycle. Histones leave the DNA only transiently during DNA replication. They stay with the DNA during transcription. –By changing shape and position, nucleosomes allow RNA-synthesizing polymerases to move along the DNA. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

51. As chromosomes enter mitosis the beaded string undergoes higher-order packing. The beaded string coils to form the 30-nm chromatin fiber. This fiber forms looped domains attached to a scaffold of nonhistone proteins. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

52. In a mitotic chromosome, the looped domains coil and fold to produce the characteristic metaphase chromosome. These packing steps are highly specific and precise with particular genes located in the same places. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Fig. 19.1

53. Chromatin

54. Interphase chromatin is generally much less condensed than the chromatin of mitosis. –While the 30-nm fibers and looped domains remain, the discrete scaffold is not present. –The looped domains appear to be attached to the nuclear lamina and perhaps the nuclear matrix. The chromatin of each chromosome occupies a restricted area within the interphase nucleus. Interphase chromosomes have areas that remain highly condensed, heterochromatin, and less compacted areas, euchromatin. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

55. DNA packing

57. Many binding sites The regulatory region of a gene is very complex in multicellular organisms Binding sites for MANY transcription factors Regulatory regions contain 1,000s of base pairs of DNA

58. DNA looping DNA is flexible and can form loops

59. DNA looping This allows transcription factors to interact over long distances

60. DNA looping Hence the RATE of transcription and the amount of mRNA and protein synthesis is determined by these interactions

61. Transcription factor binding in eukaryotes; influences The interactions of transcription factors with one another or with DNA can be influenced by signals –From within the cell –From outside the cell Signals are integrated at the promoter

62. Transcription factor—low nutrients Nutritional state of a cell may affect the activity of various transcription factors Absence of a nutrient may signal a particular repressor to bind to an operator and turn off the expression of a gene

63. Transcription factor—high nutrients For a different gene, the presence of the same nutrient may signal a transcription factor to bind in the regulator region and stimulate a high level of transcription What do we call this type of transcription factor?

64. Transcription factor—time of day At a particular time of day [lunch?] a second transcription factor may bind to augment transcription to an even higher level This type of control is an internal clock regulation and would be independent of the availability of food What do we call this type of transcription factor?

65. Transcription factor--Tissue specific Transcription of the gene that was turned on by nutrient availability and time of day in cell A may be turned off completely by the presence of a repressor in cell B Interaction of transcription factors with –DNA, other factors and the environment can dramatically alter the expression of a gene

66. Turning on a gene

67. This will cost more ENERGY! Not the best plan!

68. Post-transcriptional control Mechanism Overview These mechanisms act AFTER mRNA has been synthesized –Costs the cell more Energy! The mechanisms affect the amount of protein made by the cell

69. Tissue-specific mRNA splicing The type of proteins made by a cell can be regulated by splicing the precursor mRNA in different ways Some mRNAs are spliced in a tissue- specific manner so that two different proteins are made in two different types of cells

70. Thyroid cells splice together exons 1,2,3 and 4 of the gene to form an mRNA that is translated into the calcitonin protein

71. Brain cells splice together exons 1,2,3,5, and 6 from the same mRNA to form the neuropeptide CGRP

72. mRNA Stability The poly-A tail on mRNA is important for its stability. If the poly-A tail is removed, then RNA is rapidly degraded and less protein is made

73. mRNA stability Proteins that bind to mRNA can influence survival of the poly-A tail and hence influence the amount of protein translated form the mRNA

74. RNA compartmentalization mRNAs can be transported to specific sections of the cell in which the translated products of the mRNA will be used Some mRNAs are sequestered in sections of a cell until they are needed

75. Translational control Alters the rate at which ribosomes bind to a mRNA and make protein A tissue-specific blocking protein may bind to the RNA and inhibit the binding of ribosomes

76. Control of translation

77. Summary A gene is transcribed into mRNA in the nucleus mRNA is also processed in the nucleus by splicing and adding the poly-A tail Splicing influences the ultimate product of the mRNA IN THE CYTOPLASM, the availability of mRNA to ribosomes and the presence of molecules that either protect or degrade the mRNA will influence the amount of protein made.

78. RNA processing

80. Antibody structure Antibodies are proteins that recognize invaders [antigens] Synthesized by cells in which DNA rearrangement produces a functional antibody gene Composed of 4 polypeptide chains –2 heavy (H) & 2 light (L) –H is composed of 3 regions: variable, joining and constant: V,J & C –Each antibody recognizes and binds to one specific antigen –We need a great diversity of antibodies to protect against a variety of invaders [pathogens]

81. What are those?

82. Antibody light chain gene The genome contains several copies of the different parts of the antibody genes. In undifferentiated cells, each gene for the L of antibodies includes –100s of different V regions –Several J regions –One or more different C regions The genes for the H chains are organized similarly

84. Antibody specificity created by gene rearrangement During differentiation of immune cells that are destined to produce antibodies, the antibody genes are rearranged via recombination to produce functional antibody genes Rearrangement of the gene occurs by joining together one segment from each of the 3 regions of the gene and by deleting the other, extra DNA

85. The number of combinations of segments from the 3 regions is tremendous Which parts are exons? Which parts are introns?

86. Antibody specificity created by gene rearrangement Each L chain protein produced by the cell contains one V, J and C region The H chain is produced by a similar rearrangement process The two light chains are identical The two heavy chains are identical Each combination of a light chain and a heavy chain can recognize one specific antigen

88. 1000s of antibodies on patrol Recombination of antibody gene DNA segments can occur at many different sites but always occur between V-J and J-C regions The antibody expressed by a cell is determined by the combination of V, J and C regions that are NOT deleted. This creates the potential for each cell to make any one of 1000s of different antibody molecules, each of which can recognize a different antigen

89. Recombinations occur between V-J and J-C regions

90. Each developing antibody- producing cell has the potential to form a unique antibody Each cell makes only one type of antibody because its genes undergo rearrangement early in the differentiation of the cell The cell is thereafter committed to produce only one type of antibody

92. Gene amplification Certain regions of DNA can undergo EXTRA rounds of DNA replication

93. Gene amplification This process is called amplification

94. Gene amplification It creates MORE copies of DNA and hence the potential to make more RNA

95. Gene amplification And you know what that means… …more proteins!

96. Gene amplification of rRNA genes in amphibian eggs In amphibian eggs, genes for rRNA become highly amplified

97. Gene amplification of rRNA genes in amphibian eggs The amplification, in this case, begins with recombination between repeated copies of the rRNA genes, resulting in the production of circular DNA molecules containing the rRNA genes

98. Gene amplification of rRNA genes in amphibian eggs

99. Far more efficient that duplicating DNA the old fashioned way! These small, circular DNA molecules replicate to produce 1000s of copies of the rRNA genes This allows the egg to quickly make many ribosomes which are needed for protein synthesis after fertilization

100. Gene amplification—role in resistance to cancer drug Gene amplification can also be important in disease Methotrexate is a drug used to treat cancer patients Tumor cells that are rapidly synthesizing DNA are more damaged by the drug than normal cells are

101. Gene amplification—role in resistance to cancer drug Methotrexate inhibits the activity of dihydrofolate reductase (DHFR), an enzyme needed for synthesis of deoxyribonucleotides A cancer cell may become resistant to methotrexate if amplification of the DHFR gene occurs

102. Gene amplification—role in resistance to cancer drug Multiple copies of the gene encoding DHFR allow enough enzyme to be made for the cell to continue to grow in the presence of methotrexate

103. Gene amplification—role in resistance to cancer drug

105. Importance of gene regulation The amount and time of gene expression can be regulated at any one of several steps between the DNA and the final functional gene product