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CMMB 411- Doreen > Topic 8 > Flashcards

Topic 8 Flashcards

1
Q

What are the two major differences between DNA replication and RNA transcription?

A
  1. DNA replication copies the entire genome once and only once per cell cycle; RNA transcription selectively copies only certain parts of the genome from one to multiple times.
  2. Both DNA strands serve as templates for DNA replication; only one of the DNA strands serves as a template on which the RNA strand is built in RNA transcription.
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2
Q

In RNA transcription, which strand is used as the template?

A

The non-coding/antisense strand

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3
Q

RNA transcripts dissociate from the DNA template a few ribonucleotides behind the point of synthesis. What two things does this allow for?

A
  1. Multiple transcription of the same gene to occur at the same time.
  2. Translation to occur rapidly
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4
Q

True or false: RNA polymerase requires primers

A

False; it catalyzes RNA synthesis in the absence of primers (i.e. different that the DNA polymerase)

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5
Q

What does RNA transcription lack that is present in DNA replication?

A

Extensive proofreading mechanisms
- Still has proofreading ability, but less

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6
Q

Eukaryotes have __ polymerases, prokaryotes have __ polymerase.

A

Eukaryotes have 3 polymerases, prokaryotes have 1 polymerase.

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7
Q

RNA polymerase in all organisms contains how many subunits?

A

2 alpha and 2 beta subunits

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8
Q

Eukaryotic pol I, II, and III have ____ subunits, which are…

A

Eukaryotic pol I, II, and III have 7-11 extra subunits, which are mainly specific to each polymerase

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9
Q

What does RNA pol I transcribe in eukaryotes?

A

Large RNA (rRNA precursor)

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10
Q

What does RNA pol II transcribe in eukaryotes?

A

Protein-encoding genes

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11
Q

What does RNA pol III transcribe in eukaryotes?

A

tRNA and 5S rRNA transcription

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12
Q

What are the 3 phases of transcription?

A

Initiation, elongation and termination

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13
Q

What DNA element determines which DNA stretch undergoes transcription?

A

The promoter

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14
Q

What are the 3 steps of transcription initiation?

A
  1. Formation of a closed complex by binding a Pol to the promoter (formation of the pre-initiation complex, transcription bubble still not formed)
  2. Closed complex transformed into an open complex (i.e. transcription bubble, created by the melting of the double helices)
  3. Initial transcribing complex makes the first 10 ribonucleotides
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15
Q

Describe transcription elongation and termination

A
  • Continual RNA synthesis
  • Unwinds the DNA in the front and re-anneals it behind
  • Emergence of the growing RNA from the template
  • Proofreads

Termination: transcription stops and the RNA product is released

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16
Q

Prokaryotes require _ initiation factor(s), eukaryotes require _ initiation factors

A

Prokaryotes require 1 initiation factor, eukaryotes require several initiation factors (i.e. general transcription factors) for promoter-specific initiation

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17
Q

What 4 DNA elements are present at the core promoter?

A
  1. BRE: TFIIB recognition element
  2. Inr: Initiator
  3. TATA box
  4. DPE: Downstream promoter element
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18
Q

What proteins recognize the core promoter elements? And which elements do each of them recognize?

A
  1. TFIIB recognizes BRE
  2. TBP recognizes the TATA box
  3. TFIID recognizes the Inr and the DPE
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19
Q

How is the core promoter defined?

A

The minimal sequence needed for accurate transcription initiation in vitro

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20
Q

What is contained upstream of the core promoter?

A

Regulatory sequences required for efficient transcription in vivo

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21
Q

What are 6 examples of sequence elements contained upstream of the core promoter?

A
  1. Promoter proximal elements
  2. Upstream activator sequences (UASs)
  3. Enhancers
  4. Silencers
  5. Boundary elements
  6. Insulators
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22
Q

How do you determine the sequence of TF addition to the promoter in vitro?

A

By adding one TF at a time

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23
Q

The TBP (TATA-binding protein) is associated with…

A

~10 other TAFs (TBP-Associated Factors)

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24
Q

TFIIB binding and function

A
  • Binds the pre-initiation complex after TBP is bound
  • May function in bridging between TATA-bound TBP and Pol II
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25
Q

TFIIF binding and function

A

Recruited to promoter with pol II
- Pol II-TFIIF stabilizes the DNA-TBP-TFIIB complex and recruits TFIIE and TFIIH

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26
Q

TFIIE function

A

Recruits and regulates TFIIH

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27
Q

Largest and most complex TAF (TBP-associated factors)

A

TFIIH

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28
Q

TFIIH function

A
  • Controls ATP-dependent transition of the pre-initiation complex to the open complex
  • Phosphorylates Pol II CTD (C-terminal domain) and causes promoter melting and escape
  • Also functions in nucleotide excision repair
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29
Q

Define the pre-initiation complex

A

Protein complex containing Pol and general transcription factors.

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30
Q

Describe the recruitment of the TFIID complex, and what proteins make up the TFIID complex

A

TFIID recognizes the TATA element
- TFIID + TBP + 11 TAFs (TBP Associated factors)
- TBP (TATA- Binding protein) binds TATA box

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31
Q

How does TBP bind to the TATA box?

A
  • TBP binds the minor groove of the TATA element through its β sheet (in general, it’s the α helix: major groove binding)
  • TBP inducing induces the minor groove to undergo conformational change (widens the minor groove to almost flat and bends the DNA). This changes the topology a bit of the DNA -> opens the structure slightly
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32
Q

What binds to the promoter after TFIID binds? (2)

A

TFIIA and TFIIB

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33
Q

What is determined by the TFIIB-TBP complex?

A

Transcription direction
- It defines asymmetric assembly of the pre-initiation complex and unidirectional transcription

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34
Q

True or false: TFIIB binds the minor groove like TBP

A

True

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35
Q

Describe the structure of the CTD (carboxy-terminal domain) of Pol II of RNA polymerase II, and the reasoning for this structure

A

CTD of Pol II has repeats of Ser and Tyr for phosphorylation

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36
Q

What binds to the promoter after TFIIA and TFIIB bind? What is the structure of the DNA up to this point?

A

Pol II with TFIIF binds
- DNA is still in closed form up to this point

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37
Q

True or false: Pols I and III also have repeats for phosphorylation

A

False

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38
Q

What transcription factors bind the the pre-initiation complex after TFIIF?

A

TFIIE and TFIIH bind to complete the pre-initiation complex

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39
Q

TFIIH function after binding

A

Induces promoter melting with ATP hydrolysis and phosphorylates Pol II CTD, resulting in promoter escape

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40
Q

What occurs after TFIIE and TFIIH bind to the pre-initiation complex?

A

CTD becomes phosphorylated, promoter escape occurs, and transcription elongation begins
- The initiation stage is complete

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41
Q

What does transcription initiation require in vivo? Why?

A

Additional proteins are required because the DNA template in vivo is in chromatin form

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42
Q

What 4 additional proteins are required for in vivo transcription initiation?

A
  1. Activator proteins that recruit Pol and stabilizes Pol: promoter interaction; also binds to chromatin remodeler complexes
  2. Chromatin remodelers that modify nucleosome structure to facilitate transcription
  3. HAT: is part of the chromosome remodelling complex
  4. Mediator complex that bridges the CTD of the Pol and the activator; also regulates TFIIH
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43
Q

True or false: histone-modifying enzymes are often required for in vivo transcription initiation

A

True

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44
Q

Describe yeast and human mediators (3)

A
  • Organized in modules (i.e. sub-complexes) reflecting the different methods of isolation
  • Have similar shape and are larger than RNA pols
  • The function of most individual subunits is unknown
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45
Q

True or false: many of the mediator subunits are conserved between yeast and humans

A

True

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46
Q

Which mediator subunit is required for Pol II transcription in vivo?

A

Med17

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47
Q

Describe a technique used to study or identify components of the mediator complex

A

Immunoprecipitation followed by SDS-PAGE
To isolate protein X:
- First design anti-X antibody against “X” protein
- After immunoprecipitation, the isolated proteins are separated by SDS-PAGE, a technique that resolves proteins by size
- SDS-PAGE ca help identify proteins that interact with the protein of interest. By analyzing the gel, researchers can infer which proteins are part of the same complex.

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48
Q

____ facilitates elongation through nucleosomes

A

FACT
- protein that stands for FAcilitates Chromatin Transcription

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49
Q

What proteins make up the FACT dimer?

A
  1. Spt16:H2A:H2B
  2. SSRP1:H3:H4
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50
Q

FACT facilitates transcription elongation through…

A

Nucleosomes

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51
Q

Describe how FACT dimer facilitates elongation through nucleosomes

A

1.Spt16 disassembles H2A:H2B from the nucleosome during transcription
2. Spt16 aids in restoring H2A:H2B once the transcription bubble passes a given nucleosome

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52
Q

What happens to most initiation factors (e.g. TFIIB, TBP, etc) during transcription elongation?

A

Most initiation factors dissociate from Pol II once Pol II is activated

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53
Q

____ ______ are recruited to the CTD tail of Pol II during transcription

A

Elongation factors

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54
Q

What does the recruitment of elongation factors depend on?

A

The phosphorylation state of the CTD tail

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55
Q

Define elongation factors

A

Factors that stimulate elongation

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56
Q

ELL protein family and TFIIS elongation factors (increase/decrease) the rate of elongation by…

A

ELL protein family and TFIIS elongation factors increase the rate of elongation by limiting the time that Pol pauses
- TFIIS can also proofread the new transcript

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57
Q

What are 3 categories of RNA processing enzymes?

A
  1. 5’ capping enzymes
  2. Splicing factors
  3. 3’ polyadenylation and cleavage factors
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58
Q

What do cleavage factors do during RNA processing?

A

Cleave theRNA from the polymerase
- Frees up the RNA, post-transcriptional modifications then occur

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59
Q

Why is research on RNA so limited?

A

Because RNA is so unstable
- 5’ capping and 3’polyA tail helps with working with RNA

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60
Q

In what order are the following post-transcriptional modification enzymes recruited to the RNA transcript?
- Components of splicing machinery
-PolyA tail and cleavage factors
- Capping enzyme

A
  1. Capping enzyme
  2. Components of splicing machinery
  3. Polyadenylation and cleavage factors
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61
Q

True or false: splicing may occur before the completion of RNA synthesis

A

True
- Splicing factors may be recruited once introns are exposed

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62
Q

The RNA processing factors recruited depends on…

A

The phosphorylation state of the CTD tail

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63
Q

What are the three steps of 5’RNA cap formation?

A
  1. RNA triphosphatase removes the γ-phosphate at the 5’ end of the transcript
  2. guanyltransferase adds the GMP moiety to the terminal β-phosphate
  3. Methyltransferase adds a 7 methyl group to the guanine base
64
Q

The 5’ cap on RNA is specifically…

A

7-methylguanylate

65
Q

What are the 2 functions of the 5’ cap?

A
  1. Stabilizes the transcript
  2. Signals that the transcript is correctly processed
66
Q

What are the seven steps of 3’ polyadenylation?

A
  1. RNA Pol II encounters and transcribes the poly-A signal (usually AAUAAA)
  2. Phosphorylated CTD tail recruits polyadenylation enzymes CPSF
  3. RNA Pol II continues transcribing, but falls off shortly
  4. CPSF + CstF + other proteins cleave downstream of the poly-A signal
  5. CtsF leaves
  6. poly-A polymerase (PAP) adds ~200 adenines (A) to the 3’ end of the poly-A signal RNA
  7. poly-A binding protein (PBP) coats the As
67
Q

CstF

A

Cleavage stimulatory factor

68
Q

CPSF

A

Cleavage polyadenylation specific factor

69
Q

What are the two functions of the Poly A tail?

A
  1. Stabilizes the mRNA
  2. Signals correct 3’ end processing
70
Q

What are the two main models of transcription termination in vivo?

A
  1. Torpedo Model of Termination
  2. Allosteric Model of Termination
71
Q

Describe the Torpedo Model of Termination

A
  1. 5’ to 3’ exonuclease (torpedo) is associated with RNA pol. Torpedo = Rat1/hXrn2
  2. Once the polyA signal is exposed on the RNA transcript, the RNA transcript is cleaved from the RNA pol
  3. The torpedo exonuclease then degrades the second RNA as it has an uncapped end
  4. Degradation of the second RNA destabilizes Pol II, causing it to dissociate from the DNA template and terminate transcription.
72
Q

The allosteric model of termination is a ______-dependent model

A

Processivity

73
Q

Describe the allosteric model of termination

A
  1. RNA pol is highly processive when first transcribing the DNA
  2. Once the RNA pol transcribes the poly-A signal, the Pol II undergoes a conformational change.
  3. The conformational change of the Pol II decreases the processivity of the Pol, which reduces the affinity of the Pol for the DNA
  4. Eventually, the RNA pol dissociates
74
Q

There are (high/low) levels of Pol I transcription in cells

A

High

75
Q

Pol I is dedicated to transcribing an rRNA precursor in cells (which comes from one gene). Why do we need something with such high transcriptional activity dedicated to just one gene?

A

This rRNA gene has many isoforms (RNA transcripts from the same gene which have had different exons removed), so high transcriptional activity is required

76
Q

Describe what proteins are recruited before Pol I is recruited to transcribe the rRNA gene (3 steps)

A
  1. UBF binds first to the UCE (upstream control element)
  2. SL1 = TBP + 3 TAFs bind to the core promoter after
  3. Recruitment of RNA pol I
77
Q

What is unique about the promoters for tRNA and 5S RNA genes that are transcribed from pol III?

A
  • The promoters are internal to the tRNAs or 5S RNAs
  • Transcription starts upstream of the promoter
78
Q

Describe what proteins bind to the tRNA/5S rRNA promoter before Pol III is recruited (3 steps)

A
  1. TFIIIC binds both box A and B
  2. TFIIIB and TBP are recruited upstream of TFIIIC after TFIIIC binds
  3. Pol III is then recruited, and binsd the start site which is located between TFIIIB/TBP and TFIIIC
79
Q

What is the primary point of gene regulation?

A

Transcription initiation

80
Q

In general, transcription is controlled by…

A

Activators and repressors

81
Q

What does upstream of the core promoter contain?

A

The regulatory sequences required for efficient transcription in vivo

82
Q

Define promoter

A

Region of DNA involved in pre-initiation complex binding

83
Q

Define regulatory binding sites

A

Binding sites for different transcription factors

84
Q

Define regulatory sequences

A

The entire collection of regulator binding sites for a given gene

85
Q

Define enhancer

A

A tight cluster of regulatory binding sites that can affect long distances at either upstream or downstream of a gene

86
Q

Define the UAS (Upstream Activating Sequence)

A

An “enhancer” in yeast, only found upstream of a gene and usually not a great distance

87
Q

Define an insulator

A

Blocks promoter activation by binding activators at the enhancer

88
Q

There is (increasing/decreasing) complexity of regulatory sequences from a bacterial gene to human

A

Increasing

89
Q

Transcription activators typically contain what 2 domains?

A

Distinct DNA-binding and activation domains

90
Q

Transcription activators often act as a _____, and sometimes recognize _____

A

Transcription activators often act as a dimer, and sometimes recognize palindromic sequences

91
Q

(The same/different) activators bind to each regulatory sequence in a regulatory site; all regulatory sites (are/are not) necessarily bound at the same time

A

Different activators bind to each regulatory sequence in a genome; all regulatory sites are not necessarily bound at the same time

92
Q

What was an example of a transcription activator that we discussed in lecture and what gene does it bind?

A

Gal4, which binds the Gal1 gene in yeast

93
Q

Describe the UAS near the promoter of the Gal1 gene in yeast
- Explain how this differs from human promoters

A

Has 4 regulatory sequences, where each sequence will bind a dimer of Gal4
- Human promoters are even more complex

94
Q

What is required on the transcription activator (factor), apart from the DNA-binding domain, for eukaryotic transcription initiation?

A

An activation domain is required for eukaryotic transcription initiation

95
Q

True or false: The activating domain of a transcription factor can activate another transcription factor by binding to its DNA-binding domain (through formation of a chimera/hybrid/recombinant)

A

True

96
Q

What is a yeast two-hybrid assay in general and what is it widely used for?

A
  • A protein-protein binding assay based on properties of yeast TFs
  • Widely used to search for binding proteins through a genetic screen
  • Helps us understand protein-protein interactions
97
Q

In a yeast two-hybrid assay, what transcriptional result is observed when protein B (“prey” protein) is tethered to an activating domain, but the DNA-binding domain is absent?

A

No transcription of reporter gene

98
Q

In a yeast two-hybrid assay, what transcriptional result is observed when protein A (“bait” protein) is tethered to a DNA-binding domain but protein B with activating region is absent?

A

No transcription of reporter gene

99
Q

In a yeast two-hybrid assay, what transcriptional result is observed when protein B (“prey” protein) is tethered to an activating domain, and bound the protein A (“bait” protein)?

A

Transcription of the reporter gene

100
Q

How can His be used in a yeast two-hybrid assay

A

Put yeast in His- environment, cells will die if they don’t express His as a reporter gene -> can use to see which proteins interact to activate transcription

101
Q

Describe the “anatomy” of a DNA- binding domain (4)

A
  1. Helix-turn-helix motif
    - homeodomain proteins
  2. Zinc-containing DNA-binding domains
    - Zinc finger (e.g. TFIIIA)
    - Zinc cluster (e.g. Gal4)
  3. Leucine-zipper motif (e.g. yeast GCN4)
  4. Helix-loop-helix
102
Q

Describe the “anatomy” of activation domains (3)

A
  1. Lack of defined motifs
  2. Grouped by amino acid contents
    - e.g. Gal4 has an “acidic” activation domain
    - e.g. glutamine or proline rich
  3. May have “sticky” surfaces, often without single specificity for the protein it binds to
103
Q

Describe how the helices in helix-turn-helix motifs interact with the DNA

A
  • One helix positions the recognition helix by contacting the DNA backbone
  • The recognition helix recognizes specific base pairs in the major groove
104
Q

Homeodomain proteins have __ domains

A

3

105
Q

The recognition helix in the homeodomain proteins contains which three amino acids?

A

Ser, Arg and Asn

106
Q

How does a homeodomain contact the DNA?

A

Helix 3 contacts the major groove, while the tail from helix 1 has some additional contact the minor groove

107
Q

The zinc ion in the zinc-containing domain coordinates with which residues? What does this do?

A

the zinc ion coordinates with 2 Cys and 2 His residues, stabilizing the protein structural elements

108
Q

Describe the structure of the leucine-zipper motif

A
  • Dimerization of 2 helices that cross over one another
  • Lots of leucine near region that binds to the major groove
  • Coiled-coil domain contains precisely spaced leucine residues and olds the 2 monomers together
109
Q

Describe the helix-loop helix (HLH) protein and how it binds to the DNA

A

Dimer of 1 short alpha helix with 1 long alpha helix that bind to the major groove

110
Q

Why is Chromatin Immunoprecipitation (ChIP) used?

A

Used to determine protein-DNA interactions

111
Q

Describe the steps of ChIP for analysis of TF interactions with DNA (5 steps)

A
  1. Proteins (transcription factors) are cross-linked to DNA fragments by formaldehyde
  2. Antibody bound to magnetic beads are added to the solution of DNA fragments. this antibody is specific to the protein of interest bound the the DNA fragment
  3. Immunoprecipitate DNA-protein complex using magnet
  4. Proteins removed
  5. DNA fragment that the protein was bound to is either amplified by PCR or analyzed using a microarray (these procedures both help with sequencig the promoter region that we’re interested in)
112
Q

What types of proteins can transcription activators recruit?

A
  1. Recruit other TFs
  2. Recruits nucleosome modifiers
  3. indirectly recruits RNA pols
  4. Recruits factors needed for initiation and elongation
113
Q

How do transcription activators contribute to the complexity and diversity of eukaryotes?

A

Transcription activators work cooperatively and synergistically to promote combinatorial control, which contributes to the complexity and diversity of eukaryotes

114
Q

What specific proteins do transcription activators recruit to stimulate transcription?

A

TFIID and mediator directly, and RNA Pol II indirectly

115
Q

True or false: effects of activators are additive and/or synergistic

A

True

116
Q

The more interactinos with activators, the stronger…

A

The stimulation of transcription

117
Q

What are 2 ways that activators recruit nucleosome modifiers to improve DNA accessibility for TFs?

A
  1. Activator recruits histone acetylase that modifies histone tails and “opens up” chromatin
  2. Activator recruits proteins that use ATP to physically move nucleosomes and expose DNA (SWI/SNF family of modification complexes)
118
Q

Enhancers can act at a…

A

Great distance, and upstream or down stream of a start site (thousands of bp)

119
Q

Insulators keep enhancers from…

A

Working at a distance

120
Q

Describe the looped-out model for enhancer function and why enhancers are distance- and orientation-independent

A

In 3D, the enhancer comes in contact with the pre-initiation complex (through a TF in between) by looping towards the promoter region
- Enhancer can be upstream or downstream to loop around

121
Q

What region regulates expression of a cluster of genes?

A

The Locus Control Region (LCR)

122
Q

Give an example of a gene cluster regulated by the locus control region (LCR) in humans

A

5 hemoglobin genes exist in humans that show temporal and spatial regulation in their expression
- Expression order ε -> γ^G -> γ^A -> δ->β
- The appropriate regulators are only found in adult bone marrow (e.g. β globin is expressed in adult bone marrow and have 2 enhancers)

123
Q

How do we switch on the various globin genes in the correct order?

A

We don’t exactly know yet

124
Q

Give an example of genes regulated by LCR in mice

A
  • HoxD genes are involved in patterning the developing limbs
  • HoxD gene expression is controlled by GCR (Global Control Region) that works like LCR
  • It is not clear exactly how the LCR works, but probably works through chromatin structure
125
Q

Describe the synergistic binding of activators

A
  • The effect of activdators working together is greater than the sum of individual activator alone
  • Synergy serves as a checkpoint to ensure proper signals are receive
126
Q

Give two examples of the direct effects of activators that demonstrates synergy

A
  1. Cooperative binding through direct interaction between 2 proteins
  2. Both activators interact with a common protein by touching different parts of it
127
Q

Give two examples of the indirect effects of activators that demonstrates synergy

A
  1. First activator recruits a nucleosome remodeler to reveal a binding site a second activator
  2. Binding of first activator unwinds the nucleosome, revealing the binding site of a second activator
128
Q

Describe an example of how activators can work synergistically in S. cerevisiae for HO gene expression (signal integration)

A

SWI5, which is only active in mother cells, can recruit chromatin-remodelling complex and histone acetylase to open SBF DNA binding site up at correct cell cycle stage (G1-S transition), which allows for SBF binding to turn on HO gene transcription
- Both SWI5 and SBF are activators

129
Q

Describe an example of how activators can work synergistically in humans for β-interferon gene

A
  • β-interferon is expressed upon infection
  • architectural protein: HMGA1 (High Mobility Group A1) recruits histone modifiers
  • HMGA1 binds to the minor groove to help enhancesome assembly by keeping the enhancer straight
130
Q

What are 4 ways that transcription repressors can work?

A
  1. Block RNA pols binding site by occupying the site on the promoter (only found in prokaryotes)
  2. Interact with RNA pols at the promoter to inhibit transcription initiation
  3. Interfere with the action of activators
  4. Recruit histone modifiers to further compact the chromatin structure
131
Q

Describe how repressors can interfere with the action of activators through competition

A

If there are overlapping DNA binding sites for the activators and the repressors, the repressor can exclude the activator from binding by binding to its repressor binding site

132
Q

Describe how repressors can interfere with the action of activators through inhibition

A

Repressor binding can result in occlusion of activating region on adjacent TF

133
Q

Describe how repressors can interfere with activators, mediators or RNA pols through direct repression

A

Can bind directly and inhibit transcription machinery by interfering with activators, mediators or RNA pols

134
Q

Describe how repressors can repress expression through indirect repression

A

Repressors can recruit histone modifiers, such as deacetylase, that compact chromatin structures

135
Q

A (different/common) regulator can be found in promoters of many genes, to turn on multiple genes at the same time

A

Common

136
Q

There are (similar/different) combinations of activators/repressors specific to different cells that influence expression

A

Different

137
Q

What is a common regulator found in all three cells types in S. cerevisiae, being “a” cell, “α” cell and a/α cell (diploid)?

A

Mcm1

138
Q

What are the gene regulatory proteins in S. cerevisiae “a” cells (haploid), and what is the expression of a-specific genes, α-specific genes and haploid-specific genes?

A

Gene regulatory proteins: a1 and Mcm1

Target gene expression:
- a-specific genes: Mcm1 binds, transcription on
- α-specific genes: transcription off
- haploid-specific genes: transcription on

139
Q

What are the gene regulatory proteins in S. cerevisiae “α” cells (haploid), and what is the expression of a-specific genes, α-specific genes and haploid-specific genes?

A

Gene regulatory proteins: α1, α2 (repressor) and Mcm1

Target gene expression:
- a-specific genes: Mcm1 binds with two α2, transcription off
- α-specific genes: α1 binds with Mcm1, transcription on
- haploid-specific genes: transcription on

140
Q

What are the gene regulatory proteins in S. cerevisiae “α” cells (haploid), and what is the expression of a-specific genes, α-specific genes and haploid-specific genes?

A

Gene regulatory proteins: a1, α2 (repressor) and Mcm1

Target gene expression:
- a-specific genes: Mcm1 binds with two α2, transcription off
- α-specific genes: transcription off because α1 is not expressed
- haploid-specific genes: α2 repressor binds with a1, transcription off

141
Q

Why isn’t α1 in a/α cell (diploid) expressed?

A

Because α2 is “dominant”, which prevents α1 expression

142
Q

Describe the JAK/STAT pathway (4 steps)

A
  1. Ligand binds the extracellular domain of of JAK kinase receptor (JAK is a tyrosine kinase on the intracellular domain)
  2. Phosphorylation of receptor subunits -> causes dimerization
  3. STAT’s SH@ domain recognizes phospho-Tyr on JAK receptor, results in STAT phosphorylation
  4. STAT dimerization and DNA binding (STAT serves as a transcription factor)
143
Q

Describe the mitogen-activated protein kinase (MAPK) pathway (7 steps)

A
  1. Growth factor binds to tyrosine kinase
  2. Binding to receptor causes phosphorylation and dimerization of receptor tyrosine kinase
  3. Grb2, and adaptor with an SH2 domain, binds SOS. Grb then binds to phosphorlated domain of tyrosine kinase, which activates inactive Ras (GDP on Ras swapped out for GTP)
  4. Active Ras activates MAPKKK
  5. MAPKKK activates MAPKK which activates MAPK (MAPK is sometimes able to go into the nucleus to activate the gene target)
  6. MAPK usually activates transcription activator (e.g. Jun) by phosphorylating it
  7. Activated transcription factor enters the nucleus and binds DNA to activate transcription
144
Q

Heterochromatin is (less/more) accessible to TFs

A

Less

145
Q

What are 3 yeast telomeric sir (Silence Information Regulator) proteins? How do these proteins affect chromatin?

A
  1. Rap1 protein (DNA-binding protein)
  2. Sir2 protein (histone deacetylase)
  3. Sir 3, 4 protein
    Genes cannot be transcribed
146
Q

What are three mechanisms to block the spreading of silenced regions?

A
  1. Insulator elements can block the spread of histone modification
  2. Histone methyltransferases may repress spreading of Sir2-mediated silencing
  3. Methylation of histone H3 tail
147
Q

Methylation of H3K4 on histone H3 tail (increases/decreases) transcription

A

Increases

148
Q

Methylation of H3K9 on histone H3 tail (increases/decreases) transcription

A

Decreases
- Increases gene silencing

149
Q

True or false: methylation of histone tails always suppresses expression

A

False

150
Q

Genes can be silenced by methylation of which element of the genome?

A

Cytosines in CpG sequences

151
Q

True or false: methylation of cytosines in CpG sequences completely turn off gene expression

A

False
- There is always some leakage
- Recruitment of other proteins can turn off gene completely

152
Q

What allows for increases chromatin inaccessibility upon methylation of DNA?

A

Proteins (such as MeCP2) bind to the methylated DNA and recruit histone modifiers and nucleosome remodelers to completely turn off genes by making chromatin inaccessible

153
Q

Define imprinting

A

Parental methylation of DNA determines gene expression in the offspring

154
Q

What does imprinting ensure? Use insulin-like growth factor as an example

A

Imprinting ensures that only one parent’s copy of Igf2 is active, while H19 is expressed from the other parent. This balance is crucial for normal development.
- On the maternal chromosome, the ICR is unmethylated, allowing the protein CTCF to bind. CTCF blocks the enhancer from activating Igf2. Instead, H19 is expressed, which is involved in non-coding RNA functions and suppressing cell division.
- On the paternal chromosome, the ICR and H19 gene are methylated, which silences H19 and blocks CTCF binding. This allows the enhancer to activate Igf2 expression, which promotes growth and development.

155
Q

What is Beckwith-Wiedermann syndrome caused by? What does this result in, and what are some symptoms that the disorder is characterized by?

A

Excessive expression of paternal genes or a loss of maternal contribution of genes on chromosome 11
- Resulting in over-activity of the insulin-like growth factor 2 (Igf2) gene and/or no active copy of CDKN1C/H19 (an inhibitor of cell proliferation)

Characterized by:
- Overgrowth
- Enlarged tongue
- Midline abdominal wall defects (protrusion of umbilicus aka belly button)
- Severe hypoglycemia (low blood sugar after birth)
- Increased risk of childhood cancers

156
Q

Describe how patterns of DNA methylation can be passed on through cell divisions

A
  • CpG is palindromic, with the opposite strand also being CpG
  • Upon DNA replication, hemimethylated CpGs are methylated by a maintenance methylase

CMMB 411- Doreen (9 decks)

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