Gene Regulation in Eukaryotes

  1. In both eukaryotes and prokaryotes, transcriptional regulation occurs through the attachment of _____ ______ ______ to specific DNA sequences that are in the vicinity of the transcription unit itself. However, additional levels of complexity are both possible and necessary for controlling expression in eukaryotes for several reasons. Name 4 such reasons
    DNA-binding proteins

    • Chromatin structure often makes DNA unavailable to the transcription machinery
    • Additional RNA processing events occur
    • Transcription occurs in the nucleus, but translation takes place in the cytoplasm
    • Gene regulation needs to control cellular differentiation into hundreds of specialized cell types
  2. As in proks. gene expression in euks can be regulated at the time of transcription _________, when RNA polymerase starts to make a primary transcript. Important decisions concerning the amount of ____ ______ in the cell are indeed often made at this point
    • initiation 
    • gene product
  3. Name 5 activities in the process of gene expression that can be regulated and lead to an active gene product (excluding transcription initiation).
    • Transcript processing (including splicing)
    • export of mRNA from the nucleus
    • translatability of the message
    • localization of the protein product in specific organelles in the cell
    • modifications to the protein

    • *are all activities that can be regulated and that affect the amount of final active product
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  4. Three types of RNA polymerases transcribing genes in euks and what they regulate
    • RNA polymerase I (pol I): transcribes genes that encode the major RNA components of ribosomes (rRNAs)
    • RNA polymerase II (pol II): transcribes genes that encode all proteins
    • RNA polymerase III (pol III): transcribes genes that encode the tRNAs and certain small RNAs
  5. Although each of the regulatory regions of the thousands of pol II transcribed genes in a euk genome are unique, genes that are transcribed in a cell-type specific manner all contain two kinds of essential DNA sequences namely:
    promoters and enhancers
  6. The promoter, which is always very close to the gene's ______ ______ region, usually contains a ______ _____ (or _____ _____), consisting of roughly seven nucleotides of the sequence T-A-T-A-(A or T)-A-(A or T), located just _______ of the transcription initiation site. Binding of RNA pol to the _____ _____ allows a low, so called ____ level of transcription
    • protein coding region
    • TATA box (initiation box)
    • upstream 
    • TATA box 
    • basal level
  7. The second type of DNA sequence element important for transcription in eukaryotes is called an _______: a regulatory site that can be quite distant (10,000s of bases) from the promoter. In specific cell types, binding of proteins to enhancers can ______ or _______ basal levels of transcription
    • enhancer
    • augment or repress
  8. Enhancers may be located either ___ or ____ to the transcription start site (some are even found in ______) and a single gene can have ____ or _____ enhancers. A single enhancer may have multiple ______ _____ for different transcription factors
    Image Upload 2
    • 5' or 3' 
    • introns 
    • one or several
    • multiple transcription sites
  9. Scientists often use a gene from jellyfish that encodes green flourescent protein (GFP) as a reporter to identify enhancers in euks. How does it work? (5-story)
    • pg 549 (right middle)
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  10. Researchers can generate mutations in the control region of the GFP reporter construct and then breed transgenic animals with the ______ DNA. If GFP fluorescence is no longer seen in the same tissue, then the mutation must have affected the ______-specific enhancer. In this way, scientists can define the actual DNA sequences comprising particular ______
    • mutant
    • tissue-specific enhancer
    • enhancers
    • *Interestingly, some experiments show that enhancers can still function if their position relative to the promoter is changed (upstream or downstream)
  11. The binding of proteins to a gene's promoter and enhancer (or enhancers) controls the rate of ________ ________. Different types of proteins bind to each of the cis-acting regulatory regions: _____ factors bind to the promoter, while ______ and _______ bind to the enhancers
    • transcriptional initiation 
    • Basal factors
    • activators
    • repressors
  12. Define transcription factors

    Once the transcription factors bind to the DNA, they recruit additional ______ to the gene that can also influence _________
    Transcription factors: all sequence-specific DNA binding proteins that influence transcription (basal factors, activators or repressors)

    • proteins
    • transcription
  13. Basal factors assist the binding of RNA polymerase II to the ______. The key component of the basal factor complex that forms on most ______ is the ____ _____ _____ protein. This protein recruits other proteins called _____-_______ factors to the promoter in an ordered pathway of assembly
    • promoter
    • promoters 
    • TATA box-binding protein (TBP)
    • TBP-associated factors TAFs
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  14. Once the basal factor complex has formed, RNA polymerase can initiate a _____ level of transcription. The primary sequences and three-dimensional structures of the basal factors are highly ______ in all euks, from yeasts to humans
    • low level (basal transcription)
    • conserved
  15. Transcription of many euk genes requires a multi subunit complex called _____ that contains more than 20 proteins. _______ does not bind DNA directly but instead serves as a bridge between the ___ ____ complex at the _______, and ______ or _______ proteins bound at the enhancer
    • Mediator 
    • Mediator
    • RNA pol II
    • promoter 
    • activator or repressor
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  16. The enormous range of transcriptional regulation occurs through the binding of different ______ _____ to distinct _______ elements associated with different ______. When bound to an _____ element, transcriptional activators can interact _______ or ________ with basal factors at the promoter in a three-dimensional protein/DNA complex to cause a/an _______ in transcriptional activity
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    • transcription factors
    • enhancer elements
    • genes
    • enhancer
    • directly or indirectly
    • increase
  17. Why must DNA between the enhancer and the promoter loop for interactions to occur between basal factors, mediator and activators?
    How can enhancer sequences still function if moved to different positions relative to the promoter
    Image Upload 8
    • Because enhancers may be far removed from promoters
    • Long stretches of DNA can be quite flexible and form loops in many ways
  18. At the mechanistic level, transcriptional activator proteins bind their target sites on DNA and increase RNA synthesis by doing one or both of the following:
    pg 551 left to right
  19. Transcriptional activator proteins must bind to enhancer DNA in a _______-specific way, and after binding, they must be able to interact with other ______ (a _____ factor or a _______) to activate transcription. Name the two structural domains within the activator protein that mediate these two biochemical functions
    • sequence-specific
    • proteins (a basal factor or coactivator)
    • DNA-binding domain and the activation domain
  20. Transcription factors belong to several protein families that share similar DNA-binding domains. Two well characterized DNA-binding structures are _____ _____ _____ domains and _____ _______. Where are each found?
    • helix-turn-helix domains (mainly in euks)
    • zinc fingers (euks and proks)
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  21. DNA-binding domains fit within the _____ groove of DNA. Subtle differences in the _____ ____ sequences among activators of the same family can allow these proteins to recognize specific DNA sequence at different ______ ______
    • major
    • amino acid
    • enhancer elements
  22. The activation domains of transcription factors are less well __________ than the DNA-binding domains, and may be less ______. The amino acid sequences of activation domains depends on whether the activator interacts with the ______ complex or with one or more _________
    • characterized 
    • structured
    • basal 
    • coactivators
  23. Many activator polypeptides also have a third domain (what is it called/what does it do?)
    dimerization domain: enables them to interact with other transcription factor subunit to form multimeric proteins
  24. One structural motif in many dimerization domains is a ______ _____, a helix with leucines at regular intervals. The ability of two such _____ _____ proteins to interlock depends on the specific amino acids that lie between the ______. Most such proteins also have a characteristic DNA-binding domain, called a ______ domain
    • leucine zipper
    • leucine zipper protiens
    • leucines
    • basic domain
  25. Among the best-characterized transcripton factors with a leucine zipper is Jun, a protein important for cellular proliferation and other processes such as the ____ and _______ of the uterine endometrium during the mammalian menstrual cycle. Jun can form dimers either with itself, making Jun-Jun ________, or with other proteins called Fos, making Jun-Fos ________
    • loss and regeneration
    • homodimers
    • heterodimers
    • *check the tape
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  26. Repressors
    Euk transcription factors that bind specific DNA sites near a gene, such as enhancers, and prevent the initiation of transcription of the gene
  27. In prokaryotes, negative regulators generally work by ______ ______ RNA polymerase from binding the promoter. In euks, the primary function of repressors in different (explain)
    • physically blocking 
    • Euk repressors generally recruit corepressor proteins to enhancers. Corepressor proteins on their own cannot bind to DNA, so they can associate with enhancers only if a repressor with which they can associate is already there
  28. What are the two possible functions of corepressors?
    Image Upload 11
    pg 552
  29. How are repressor structures similar to activators?
    • Repressors have DNA-binding motifs, repression domains for interacting with corepressors, and some have dimerization domains.
    • In fact, certain transcription factor can act as either activators or repressors, depending on context
  30. The drosophila transcription factor called Dorsal is an activator when bound to the enhancers of some target genes and a repressor when bound to the enhancers of other genes. How can one protein have two opposing functions in transcription?
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    Dorsal is intrinsically an activator that binds coactivators. However, at some enhancers, interactions with another protein causes Dorsal to bind instead a corepressor called Groucho
  31. Many regulatory proteins, called ______ _______, prevent transcription initiation indirectly, not by recruiting corepressors, but instead by interfering with the function of activators. Name the four ways this is accomplished
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    • indirect repressors
    • pg 553 left and right
  32. Scientists often use GFP reporter genes to verify that a transcription factor suspected of regulating the expression of a target gene indeed plays such a role. What are the two requirements of this experimental approach?
    pg 553 right
  33. By contrast, loss of function mutations in a gene encoding a repressor may yield _____ levels of GFP flourescence than normal, and/or the expression of GFP in _________ cells
    • higher levels
    • inappropriate cells
  34. In complex multicellular organisms, a large percentage of genes are devoted to transcriptional regulation. Of the 25,000 genes in the human genome, scientists estimate that more than _____ of them encode transcription factors. Many proteins can regulate the ______ of any one gene, and each regulatory protein may act on ______ different genes
    • 2000
    • transcription
    • many 
    • *The number of possible combos of regulators is staggering and provides the flexibility important for differentiation of cells and development in multicellular euks
  35. Operational definition of an enhancer
    a cis-acting sequence that controls a gene's expression in a particular type of cell at a particular moment in time
  36. Why is it that a gene's regulatory region may contain several enhancer elements, such as an eye enhancer that activates transcription in eye cells and a skin enhancer that activates transcription in skin cells
    • because many genes are expressed in more than one tissue
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  37. Each enhancer in turn has one ore more binding sites with varying _______ for each of several different activators and repressors. At any moment, there may be dozens of _____ and ________ in the cell that compete for these binding sites, as well as _______ and _______ that compete with each other for binding to different ________ or ________. All this info biochemically integrated from an enhancer guides the cell to do what two things?
    • affinities
    • activators and repressors
    • coactivators and corepressors 
    • activators and repressors
    • 1)Decide whether a gene should be turned on or off
    • 2)helps the cell to fine-tune a gene's transcriptional activation or repression to a level optimal for the cell's role in the organism
  38. Within a multicellular organism, all cells have the same ______ _____, so a gene's enhancer elements are present in ____ types of cells. The cell-type specificity of transcription therefore depends on changes that occur during which process?
    • nuclear DNA
    • all
    • the course of development to the constellation of transcription factors that interact with these enhancers
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  39. Euk transcription factor activity can be regulated through allosteric interactions with small molecules. Steroid hormone receptor transcription factors constitute an important exmaple of allostery in euks. (explain the example)
    • Hormone binding causes an allosteric change in the receptor protein that greatly increases the affinity of its DNA-binding domain for its target enhancer
    • *In humans, the steroid hormones testosterone and dihydrotestosterone work precisely through this mechanism
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  40. Transcription factor proteins can be modified after they are synthesized by the ______ addition of any of several different chemical groups. One of the most important of these modifications is phosphorylation (define)
    • covalent
    • the addition of a phosphate group to a protein by action of an enzyme called a kinase
  41. Phosphorylations can either ______ or ______ a transcription factor in any of a number of ways. List 4
    • activate or deactivate 
    • pg 555 left
  42. Cells often rely on phosphorylations to control events that must occur _______ (example)
    • rapidly
    • Ex: such as repsonses to changes in the environment or transitions between states in the cell cycle
  43. Not all transcription factors are made in ____ cells at ____ times. Clearly, if a factor is not present in a given cell, it will be ______ to influence the initiation of the ________ of any target genes. (explain)
    • all 
    • all
    • unable
    • transcription 
    • In other words, the availability of various transcription factors is critical to a cell's determination of which genes will be transcribed and if so, at what levels
  44. An enhancer may be located ______ or ______ of the promoter that it regulates and in either orientation with respect to the promoter. These facts pose a conceptual problem. Pg 555 top right
    the DNA elements called insulators organize chromatin so that enhancers have access only to particular promoters
  45. Insulators (define)
    How do we test if something is an insulator in a reporter gene?
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    • DNA elements located between a promoter and an enhancer that block the enhancer from activating transcription from that promoter. 
    • Suspect insulator DNA sequences are inserted between an enhancer and a promoter of a reporter gene; if reporter gene expression is blocked then the DNA sequence is deemed an insulator
  46. How insulators work (2-story)
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    pg 555 right
  47. Cis acting elements
    Trans acting elements
    • Cis acting elements: Elements that are on the same chromosome as the gene being affected. DNA elements.
    • Trans acting elements: Elements that do not need to be on the same chromosome as the gene being affected. Protein elements.
  48. chromatin immunoprecipitation sequencing
Card Set
Gene Regulation in Eukaryotes
Ch 16.1-16.3