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BIOM 3500 - Midterm 2: Regulation of Gene Expression > Lecture 9 and 10 - Regulation of Transcription > Flashcards

Lecture 9 and 10 - Regulation of Transcription Flashcards

1
Q

Why do cell types in a multicellular organism become different from one another?

A

Because they synthesize and accumulate different sets of RNA and protein molecules without altering the sequence of their DNA

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2
Q

What is the main difference for why genes with the same DNA are different from one another?

A

Transcriptional regulation

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3
Q

What happens when the nucleus of a fully differentiated frog cell is injected into a frog egg whose nucleus has been removed?

A

The injected donor nucleus is capable of directing the recipient egg to product a normal tadpole which contains a full range of differentiated cells that derived their DNA sequences form the nucleus of the original donor cell. Thus, the differentiated donor cell cannot have lost any important DNA sequences.

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4
Q

What happens when the nucleus of a fully differentiated frog cell is injected into a frog egg whose nucleus has been removed?

A

The injected donor nucleus is capable of directing the recipient egg to product a normal tadpole which contains a full range of differentiated cells that derived their DNA sequences from the nucleus of the original donor cell. Thus, the differentiated donor cell cannot have lost any important DNA sequences.

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5
Q

What happens when the nucleus of a fully differentiated frog cell is injected into a frog egg whose nucleus has been removed?

A

The injected donor nucleus is capable of directing the recipient egg to produce a normal tadpole which contains a full range of differentiated cells that derived their DNA sequences from the nucleus of the original donor cell. Thus, the differentiated donor cell cannot have lost any important DNA sequences.

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6
Q

How is RNA data obtained by the RNA-seq technique?

A

RNA is collected from cell lines grown in culture and “sequence reads” are obtained and mapped across the human genome by matching RNA sequences to the DNA sequence of the genome. At each position along the genome, the height of the coloured trace is proportional to the number of sequence reads that match the genome sequence at that point. Exons are transcribed genes present in high levels, reflecting their presence in mature mRNAs. Intron sequences are present at much lower levels and reflect pre-mRNA molecules that have not yet been spliced plus intron sequences that have been spliced out but not yet degraded.

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7
Q

What does it mean if the RNA seq from cells are almost never sequenced? (No large peaks)

A

Other cells don’t need the protein so they don’t make the transcript

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8
Q

Why are genes only transcribed in some cells/at specific times?

A
  • Gene specific transcription activators are present in specific cell types at specific times
  • Gene specific transcription activators interact (indirectly) with RNA polymerase II/Transcription factor IIs to strengthen their interaction with the promoter
  • They also remodel chromatin at the promoter (removal of histones) to allow access of the promoter to RNA polymerase II/Transcription factor IIs
  • Gene specific receptors have opposing effects
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9
Q

How can a cell control the proteins it makes?

A

1) Transcriptional control
2) RNA processing control
3) RNA transport and localization control
4) Translational control
5) mRNA degradation control
6) Protein activity control

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10
Q

For most genes, why are transcriptional controls paramount?

A

Because out of all the possible control points, only transcriptional control ensures that the cell will not synthesize superfluous intermediates

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11
Q

What are transcription regulators?

A

Proteins that recognize specific sequences of DNA (typically 5-10 nucleotide pairs in length) that are often called cis-regulatory sequences, because they must be on the same chromosome to the genes they control

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12
Q

What happens when transcription regulators bind to cis-regulatory sequences dispersed throughout the genome?

A

Puts into motion a series of reactions that ultimately specify which genes are to be transcribed and at what rate

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13
Q

What are cis-regulatory sequences also known as?

A

Enhancers or enhancer elements

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14
Q

What may regulatory sequences be bound to?

A

Transcriptional activators and/or repressors - these sequences and the proteins that bind to them are gene specific

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15
Q

What is the combined effect of regulators?

A

To promote or inhibit RNA polymerase binding to the promoter

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16
Q

What about the studded DNA sequence information do the transcription regulators recognize?

A

The edge of the base pairs that represent a distinctive pattern of hydrogen-bond donors, hydrogen-bond acceptors, and hydrophobic patches in both the major and minor grooves

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17
Q

Why do the majority of transcription regulators make contact with the major groove?

A

It’s wider and displays more molecular features than the minor group

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18
Q

Why does a transcription regulator recognize a specific cis-regulatory sequence?

A

Because the surface of the protein is extensively complementary to the special surface features of the double helix that displays that sequence

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19
Q

Why are cis-regulatory sequences often depicted as “logos”?

A

Sequence-specific DNA-binding recognize a range of closely related sequences with the affinity of the protein for the DNA carrying according to how closely the DNA matches the optimal sequence

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20
Q

What does the arrangement of forming dimers of transcription regulators do?

A

Doubles the length of the cis-regulatory sequence recognizes and greatly increases both the affinity and the specificity of transcription regulator binding - causes many fewer random occurrences of matching sequences

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21
Q

What are heterodimers often formed from?

A

Two different transcription regulators

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22
Q

When transcription regulators form heterodimers with more than one partner protein, what can be “reused?”

A

The same transcription factor to create several distinct DNA-binding specificities

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23
Q

What do most transcription factors form?

A

Homodimers or heterodimers - Most transcription factors will not activate transcription unless bound as dimers

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24
Q

What families do most transcription factors fall into?

A
  • Helix-turn-helix
  • Homeodomain
  • Leucine zipper
  • Zinc finger
  • Helix-loop-helix
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25
Q

What are homeodomain proteins?

A

Proteins folded into three alpha helices with helix 3 being able to form important contacts with the major groove of DNA, which can only interact if the proper sequence within the transcription factor is there

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26
Q

What are helix-turn-helix proteins?

A

Proteins with motif constructed from two alpha helices with the more C-terminal helix called the recognition helix, capable of fitting into the major groove of DNA, playing an important part in recognizing the specific DNA sequence to which the protein bind

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27
Q

What is a leucine zipper protein?

A

Protein where two alpha helices, one from each monomer, are joined together to form a Y-shaped structure, allowing their side chains to contact the major groove of DNA

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28
Q

What are zinc finger proteins?

A

Group of DNA-binding motifs with one or more zinc atoms found in clusters with the alpha helix of each finer contacting the major groove of the DNA

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29
Q

What do members of a family share and what do they differ in?

A

Share a common structure but differ in the specific amino acid that contacts base pairs in the DNA

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30
Q

What does each transcription recognize?

A

A specific motif (each transcription factor may recognize multiple sites in the genome)

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31
Q

How are transcription factors affected by binding sites?

A

They may vary and have different affinities for the transcription factor

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32
Q

Why does cooperative binding of transcription regulators to DNA often occur?

A

Because the monomers have only weak affinity for each other

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33
Q

Why do transcription regulators bind to DNA in nucleosomes with lower affinity than they do to naked DNA?

A

1) Surface of cis-regulatory sequence recognized by the transcription regulator may be facing inward on the nucleosome, toward the histone core, and therefore not readily be available to the regulatory protein
2) Many transcription factors subtly alter the conformation of the DNA when they bind, and these changes are generally opposed by the tight wrapping of the DNA around the histone core

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34
Q

How do co-activators promote RNA polymerase binding to promoter?

A

1) Indirectly - Co-activators bind to histone remodeling proteins, histone-modifying enzymes
–> Displacement/modification of histones opens up promoter to allow RNA polymerase binding
2) Directly - Co-activators also bind to RNA polymerase (via Mediator complex) which helps to stabilize/recruite RNA polymerase binding to promoter

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35
Q

What modifications of local chromatin structure favour transcription initiation?

A
  • Nucleosome remodeling
  • Nucleosome removal
  • Histone replacement
  • Certain types of covalent histone modifications
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36
Q

What do eukaryotic transcription activators attract?

A

Coactivators that include histone modification enzymes, ATP-dependent chromatin remodeling complexes, and histone chaperones, each of which can alter the chromatin structure of promoters

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37
Q

How do eukaryotic transcription activator proteins direct local alterations in chromatin structure?

A
  • Nucleosome sliding allows access of transcription machinery to DNA
  • Transcription machinery assembles on nucleosome-free DNA
  • Histone variants allow greater access to nucleosomal DNA
  • Specific patterns of histone modification destabilize compact forms of chromatin and attract components of transcription machinery
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38
Q

What do alterations in chromatin structure increase and facilitate?

A

Increases the accessibility of DNA and facilitates the binding of RNA polymerase and the general transcription factors

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39
Q

In general, what do HATs and histone demethylases recruited from transcription activators do?

A
  • Histone acetylation via histone acetyl transferase (HATs) opens chromatin to promote RNA polymerase binding
  • Histone methylation via histone methyl transferases promotes closed chromatin configuration to prevent RNA polymerase binding
  • Histone deacetylases and histone demethylases can reverse these moditifactions
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40
Q

What does the stepwise assembly of the general transcription factors at a eukaryotic promoter provide (in principle)?

A

Multiple steps at which the cell can speed up or slow down the rate of transcription initiation in response to transcription regulators

41
Q

What is the term, gene control region, used to describe?

A

The whole expanse of DNA involved in regulating and initiating transcription of a eukaryotic gene

42
Q

What does the gene control region include?

A

The promoter where the general transcription factors and the polymerase assemble, plus all of the cis-regulatory sequences to which transcription regulators bind to control the rate of the assembly processes at the promoter

43
Q

Where can cis-regulatory sequences be located?

A

Adjacent to the promoter, far upstream of it, or even within introns or entirely downstream of the gene

44
Q

What do the broken stretches of DNA in the gene control signify?

A

That the length of DNA between cis-regulatory sequences and the start of transcription varies, sometimes reaching tens of thousands of nucleotide pairs in length

45
Q

What does DNA looping allow for?

A

Allows transcription regulators bound at any of the positions to interact with the proteins that assemble at the promoter

46
Q

What do transcription regulators act through?

A

Many act through the mediator, some interact with the general transcription factors and RNA polymerase directly, and others recruit proteins that alter the chromatin structure of the promoter

47
Q

What can transcription repressors do?

A

Depress the rate of transcription below the default value and rapidly shut off genes that were previously activated

48
Q

What do eukaryotic repressor mechanisms ultimately do?

A

Block transcription by RNA polymerase, typically by bringing co-repressors to DNA

49
Q

In what ways do eukaryotic repressor proteins operate?

A

1) Competitive DNA binding - Activator proteins and repressor proteins compete for binding to the same regulatory DNA sequence
2) Masking the activation surface - Both proteins bind DNA, but the repressor prevents the activator from carrying out its functions
3) Direct interaction with the general transcription factors - The repressor blocks assembly of the general transcription factors
4) Recruitment of chromatin remodeling complexes - The repressor recruits a chromatin remodeling complex, which returns the nucleosomal state of the promoter region to its pre-transcriptional form
5) Recruitment of histone deacetylases - Repressor attracts a histone deacetylase to the promoter which can reverse transcription initiation
6) Recruitment of histone methyl transferase - Repressor attracts a histone methyl transferase, which modifies certain positions on histones by attaching metyl groups

50
Q

How can transcription repression be especially efficient?

A

By acting through more than one mechanism at a given target gene

51
Q

What keeps a transcription regulator bound on the control region of one gene from looping in the wrong direction and inappropriately influencing the transcription of an adjacent gene?

A

Several types of DNA elements compartmentalize the genome into discrete regulatory domains

52
Q

What do barrier sequences do?

A

Prevent the spread of heterochromatin into genes that need to be experessed

53
Q

What is an insulator?

A

A type of DNA element that prevents cis-regulatory sequences from running amok and activating inappropriate genes

54
Q

How do insulators function?

A

By forming loops of chromatin which hold a gene and its control region in rough proximity and help to prevent the control region from “spilling over” to adjacent genes

55
Q

In simple terms, what do insulators, barrier sequences, and insulator-binding proteins do?

A
  • Insulators directionally block the action of cis-regulatory sequences, whereas barrier sequences prevent the spread of heterochromatin
  • Insulator binding proteins hold chromatin in loops, thereby favouring “correct” cis-regulatory sequence-gene associations
56
Q

What was the experiment for the identification of a boundary (insulator) element?

A
  • Transposable element (TE) containing white gene coding sequence and partial enhancer –> low level transcription of the gene
  • Allow TE to randomly insert into genome
  • If it inserted far from any other gene –> low level expression of gene
  • If it is inserts near a strong enhancer –> higher level expression of gene
  • scs/scs’ domains protect sequence within from position effects and only work when flanking the gene (a single element does not work)
  • scs sequence binds a protein: Zw5
  • scs’ sequence binds a protein: BEAF
  • GST-Zw5 pulls down bacterially purified BEAF
57
Q

What happens once a cell in a multicellular organism becomes committed to differentiate into a specific cell type?

A

The cell maintains this choice through many subsequent cell generations, which means that it remembers the changes in gene expression involved in the choice

58
Q

What happens during the development of the Drosophila egg from fertilization to the cellular blastoderm stage?

A

1) Fertilized egg
2) Many nuclei divide rapidly in a syncytium
3) Nuclei migrate to the periphery, where cell boundaries will eventually form (only then do cell membranes start being made and separate off)

59
Q

What does the embryo in a single giant cell contain at the stage of development when Eve begins to be expressed?

A

Multiple nuclei in a common cytoplasm which contains a mixture of transcription regulators that are distributed unevenly along the length of the embryo, thus providing positional information

60
Q

What does positional information distinguish?

A

One part of the embryo from another

61
Q

Why do initially identical nuclei rapidly begin to express different genes?

A

Because they are exposed to different transcription regulators

62
Q

How is Drosophila segmented in the embryo?

A
  • No segments are visible at first but a fate map showing the future segmented region is shown
    –> Head parts, thorax, abdomen
  • Segments are then clearly defined
63
Q

How was the identification of patterning genes in Drosophila discovered?

A

Wieschaus and Nusslein-Volhard did a genetic screen for Drosophila embryonic lethal mutants to identify those that arrest prior to embryo hatching and that appear normal in some parts of the embryo but abnormal in other parts (Nobel prize in 1995)

64
Q

According to their mutant phenotypes, what three groups do segmentation genes fall into?

A

1) Gap genes - eliminates one or more groups of adjacent segments
2) Pair-rule genes - cause a series of deletions affecting alternate segments, leaving the embryo with only half as many segments as usual
3) Segment-polarity genes - produce normal number of segments but with a part of each segment deleted and replaced by a mirror-image duplicate of all of part of the rest of the segment

65
Q

What have the regulatory DNA sequences that control the Eve gene evolved to do?

A

“Read” the concentrations of transcription regulators at each position along the length of the embryo causing the Eve gene to be expressed in seven precisely positioned stripes, each initially 5-6 nuclei wide

66
Q

What is the regulatory region of the Eve gene formed from?

A

A series of relatively simple regulatory modules, each of which contains multiple cis-regulatory sequences and is responsible for specifying a particular stripe of Eve expression along the embryo

67
Q

How was the modular organization of the Eve gene control region revealed?

A

By experiments in which a particular regulatory module say, that specifying stripe 2, is removed from its normal setting upstream of the Eve gene, place in front of a reporter gene, and reintroduced into the Drosophila genome

68
Q

Describe the steps of the experiment demonstrating the modular construction of the Eve gene regulatory region.

A

1) A 480-nucleotide-pair section of the Eve regulatory region was removed and inserted upstream of a test promoter that directs the synthesis of the enzyme beta-galactosidase
2) When this artificial construct was reintroduced into the genome of the Drosophila embryos, the embryos expressed B-galactosidase precisely in the position of the second of the seven Eve stripes

69
Q

What does beta-galactosidase serve as and why?

A

Serves as a reporter since it “reports” the activity of a gene control region and it is simple to detect, thus providing a convenient way to monitor the expression specified by a gene control region

70
Q

What is Bicoid (Bcd)?

A
  • A homeodomain transcription factor that promotes the transcription of genes required for anterior fates (can also bind RNA and regulate translation)
  • The egg-polarity gene responsible for the signal that organizes the anterior end of the embryo
71
Q

Describe the Bicoid protein gradient

A

1) Bicoid mRNA is deposited at the anterior pole during oogenesis
2) Local translation followed by diffusion generates protein gradient
- Diffuses away from its source with its large concentration remaining at the head of the embryo

72
Q

How was the Bicoid protein gradient discovered?

A

RNA in situ hybridization and immunolocalization
- Detects DNA or RNA sequence by taking a labeled complementary sequence and having it hybridize
- Add Bicoid DNA or RNA that can be detected

73
Q

What other expression does the Bicoid gradient determine (Bcd targets)?

A

Patterns of Hunchback (Hb), giant, and orthodentical gene (Otd) required in the embryo *zygotic genes

74
Q

What special feature do egg-polarity genes have?

A

They are all maternal-effect genes, in that it is the mother’s genome rather than the zygote’s genome that is critical

75
Q

What happens to a fly’s offspring if they have mutant chromosomes for Bicoid?

A

Male flies will develop normally but the female offspring won’t be able to deposit any functional Bicoid mRNA into her own eggs so they will develop into headless embryos

76
Q

What is the Hunchback gene concerned with?

A

The expression in the anterior half of embryos

77
Q

Why do Hb and Otd genes produce different patterns when affected by the Bcd gradient?

A

The have different binding sites and affinity for Bcd
- Otd has 2 weak sites and it’s pattern is seen on the anterior side
- Hb has 3 weak sites and 3 strong sites and it’s pattern is seen largely on the anterior side and partly on the posterior side

78
Q

What are the two transcription regulators that activate Eve transcription and what are the two transcription regulators that repress it?

A

Activators: Bicoid and Hunchback
Repressors: Krüpel and Giant

79
Q

How is Giant seen to be expressed?

A

Expressed at anterior end and then repressed by another transcription factor but then activated by another transcription factor near the posterior side so it becomes expressed again

80
Q

How is Krüpel seen to be expressed?

A

Repressed by Hb at anterior side so it’s expressed in the middle and then gets repressed by another repressor present at the posterior end

81
Q

What do the relative concentrations of the four proteins involved in regulating the Eve stripe 2 do?

A

Determine whether the protein complexes that form at the stripe 2 module activate transcription of the Eve gene

82
Q

What will turn off the stripe 2 module?

A

Either of the two repressor proteins bound to the DNA

83
Q

What will create maximal activation of Eve stripe 2?

A

Both Bicoid and Hunchback binding

84
Q

What must the conditions be in order for the stripe 2 module to be turned on and therefore express the Eve gene?

A

The levels of both Bicoid and Hunchback must be high and both Krüpel and Giant must be absent

85
Q

Where does the combination of present Bicoid and Hunchback and absent Krüpel and Giant occur in the embryo?

A

Occurs only in one region of the early embryo

86
Q

Although it is not known exactly how the 4 transcription regulators interact with coactivators and co-repressors to specify the final level of transcription across the stripe, what does the outcome very likely rely on?

A

The competition between activators and repressors when binding sites for the transcription regulators can be seen to overlap

87
Q

How were the distributions of the transcription regulators discovered to be responsible for ensuring that Eve is expressed in stripe 2?

A

Distributions of these proteins were visualized by staining a developing Drosophila embryo with antibodies directed against each of the four proteins

88
Q

In fly embryos that lack Krüpel, what happens to stripe 2?

A

Expands posteriorly

89
Q

In fly embryos that have mutated binding sites for Krüpel, what happens to stripe 2?

A

Expands posteriorly because the DNA-binding sites will be inactivated by mutation

90
Q

If Giant is absent, how would the Giant mutant be expressed?

A

It would be longer along the anterior site because, in theory, Giant has to be binding to the DNA of the Eve stripe 2 regulatory sequences in order to repress transcription

91
Q

How can you determine DNA sequences that a transcription factor binds to?

A
  • Old method: DNase footprinting
  • Newer method: Chromatin IP (ChIP)
92
Q

How is chromatin-IP followed by DNA sequencing (Chip-seq) carried out?

A

Start with transcription regulator A on gene 1 and transcription regulator B on gene 2
1) Add cross-linker to cells (e.g., formaldehyde)
2) Proteins bind to nearby proteins or DNA
3) Lyse cells
4) Break DNA into small fragments
4) Immunoprecipitate transcription factor by using antibodies against transcription regulator A
5) Reverse formaldehyde cross-links to elute DNA; remove protein
- Determine the sequence of DNA

93
Q

What does the number of ChIP sequence reads indicate?

A

The frequency of protein occupancy - relates to affinity and protein concentration

94
Q

What did Giant ChIP reveal?

A

3 sites upstream of Eve gene and within Eve stripe 2 enhancer

95
Q

What would the expression of the embryo be like if there were mutations in all 3 Giant binding sites?

A

It would behave like a Giant mutant and the stripe would be longer anteriorly

96
Q

What is Krüppel repressed by?

A

Hunchback (only expressed when Hb levels are low)

97
Q

How are the three different domains of Eve strip 2 expressed of repressed?

A

1) Bicoid and Hunchback are present at high levels but Giant binds to its binding site and represses the transcription of Eve anteriorly
2) Bicoid and Hunchback are present without the presence of Giant so the stipe forms
3) Krüpel binds and represses Hunchback and the transcription of Eve stripe 2 is repressed posteriorly (Krüpel may also indirectly interfere with Bicoids transactivation activity)

98
Q

Summarize the binding of proteins involved in the transcription of Eve stripe 2

A
  • Has binding sites for Bcd, Hb, Giant, and Krüpel
  • Bcd and Hb must be bound for Eve to be transcribed
  • Bcd sites are low-affinity –> synergistic effect on RNA polymerase recruitment
  • Giant and Krùpel binding can prevent the binding of Bcd and Hb, or block their interaction with RNA polymerase II
  • Eve gene has other binding sites for other transcriptional regulators found in other parts of the egg

BIOM 3500 - Midterm 2: Regulation of Gene Expression (4 decks)

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