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BIOL 200 > Protein regulating transcription-activators (RR5-) > Flashcards

Protein regulating transcription-activators (RR5-) Flashcards

1
Q

TFIIH required for pol I and pol III as well as for pol II?

A

No, RNA pol I and RNA pol III do not have ATP-dependent helicase step, can do denaturing themselves
(pol I and III also do not have CTD tail to be phosphorylated)

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2
Q

Does TFIID act on all DNA? even the ones with out a TATA-box?

A

Yes, TBP still binds to form a kink
TAF (TBP associated factors) interact with enhancers proximal to transcriptional start site

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3
Q

How does the Electrophoresis mobility shift assay (EMSA) work? What is it useful for?

A

Assess for DNA binding activity
Needs Probe = radiolabelled dsDNA
1. Run liquid chromatography and retain multiple fraction of the solution (each fraction has different composition, heavier proteins in first samples, light in last fractions)
2. Make radiolabelled dsDNA interact with every fraction (with all proteins)
3. Run non-denaturing polyacrylamide gel with every fraction as a column
Proteins binding to DNA shifts the mobility of DNA in the gel (runs slower if bound)

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4
Q

How can you use recombinant vectors to test DNA binding transcription factors?

A

Plasmid 1 = cDNA of protein of interest
Plasmid 2 = X binding site (controlled element) + lacZ reporter gene
(Plasmid 3 = cDNA of a protein that would act as co-factor to protein of interest if want to test cooperativity)

Co-Transfection of plasmids into eukaryotic cell, if expression enhanced, then binding of protein of interest happened, if not enhanced, protein of interest didn’t bind the site

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5
Q

What is Helix recognition?

A

Recognition of a DNA site by a alpha-helical domain by interaction with MAJOR groves by non-covalent interactions

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6
Q

What does it mean for transcription factors to be modular?

A

Most transcription factors have multiple domains that each perform distinct functions

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7
Q

How does GAL4 transcription factor from yeast expresses its modular

A

DNA binding domain = C6 Zinc finger domain
- DNA binding domain to bind UASgal (at start of UAS/ N-terminus)
- Activation domain to stimulate transcription (at end of UAS/ C-terminus)
Test by taking short sequences out by linker scanning mutations

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8
Q

What are the possible domains transcription factors can possess?

A
  • DNA binding domain
  • Transcription activating domain
  • Transcription repression domain
  • Chromatin remodelling domain
  • nuclear import domain
  • protein interaction domain

*Transcriptional activating domain seems to be similar in different TF so could be interchangeable

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9
Q

What are Homeodomain proteins?

A
  • Presence in several transcription factors giving rise to homeotic transformations when mutated at particular residues
  • Involved in spacial and temporal patterns of gene expression
  • Binds to homeoboxes (specific DNA sequences in promoter regions of target genes), Hox genes
  • Helix-loop-helix structure (DNA binding)
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10
Q

What are the different types of zinc finger DNA binding domains?

A
  • C2H2 types: 2 cysteine + 2 Histones, 3+ finger units (3+ times C2H2 each bind to a Zin++) and bind to DNA as monomer (only zinc finger motif, no cooperativity with other TF)
  • C4 types: 4 Cysteines, 2 finger units (2 Zn++), binds to DNA as homo or heterodimers (streroid hormone receptors)
  • C6 types: 6 Cysteines metal ligands coordinately bind to 2 Zn2+ ions (ex: GAL4)
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11
Q

In what form/combinations do Leucine zipper proteins bind to DNA?

A

They bind to DNA as homo- heterodimers

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12
Q

What is the structural particularity of Leucine zipper proteins that allows it to bind to DNA?

A

They contain a leucine or other hydrophobic amino acid in heptead repeats in the C-terminal region of the DNA binding domain
These hydrophobic residues form a dimeric coiled coil domain required for dimerization
The coiled coil motif if followed by extensions of the alpha-helices (1 from each leucine zipper) that interacts with the major groves of DNA

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13
Q

What is the difference between leucine zipper proteins and Helix-Loop-Helix proteins (2 classes of TF)?

A

Leucine zipper = 1 extended alpha-helix
HLH = 2 alpha-helices connected by short loop

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14
Q

How is qualified the binding of TF of unrelated classes working together? In what cases is it important?

A

They bind cooperatively
If two proteins bind weakly to DNA, enhancing transcription very weakly, as they cooperate, they can bind more strongly and have a greater enhancing effect
ex: If there are 2 weak binding site next to each other, cooperative binding can make it a strong binding site

Cooperative binding can be on or off the DNA

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15
Q

What is the structure of Helix-Loop-Helix DNA binding proteins? (Transcription Factors)
And How do they dimerize?

A
  • 2 Helices: N-terminal helix and C-terminal helix, connected by a short loop
  • Loop region connects 2 alpha-helices, does the interaction with other proteins, stabilization of the binding and dimerization, etc.
  • N-terminal alpha-helix recongizes specific DNA by interaction with major grooves (non-covalently)
    Some amino acids H-bond to DNA bases
  • Dimerization: C-terminal helices of both HLH interact in coiled coil motif fashion as they have hydrophobic amino acids at intervals characteristic of an amphipathic alpha-helix
  • Homodimer = HLH of 2 proteins of same nature dimerize
  • Heterodime = HLH of different proteins
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16
Q

What aspect of DNA binding extends the potential for diversified gene regulation?

A

Cooperative binding / Combinatorial possibilities

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17
Q

How is the sequence of DNA bound to TF called?

A

Promotors

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18
Q

How do we call the proteins that bind to promotors to enhance or repress transcription activity?

A

Transcription factors

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19
Q

What happens when 3 different TF homo- or heterodimerize?

A

Gives rise to 6 different combination / 6 different possible binding sites on different promotor sequence that have more or less influence
More precise regulation of transcription

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20
Q

What method is used to identify the DNA sequence of the segment the TF binds to?

A

ChIP-Chromatin Immunoprecipitation (ChIP-seq)

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21
Q

What is the proceedure of ChIP - Chromatin Immunoprecipitation?

A
  1. Crosslink macromolecule with formaldehyde (form covalent bond between DNA and its protein)
  2. Shear DNA into small fragments (some fragments bound to protein of interest, some not)
  3. Immunoprecipitate with an antibody for the protein of interest to have a sample with only the fragments of DNA + protein
  4. Purify DNA
  5. Next-Gen sequencing of DNA
  6. DNA will correspond
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21
Q

Which Pol has more sensitivity to a-amanitin?

A

pol II = most sensitive to alpha-amanitin
pol I = not sensitive
pol III = intermediate sensitivity

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22
Q

What is the role of the Mediator complex?

A

It bridged vast sections of chromatin to enhance transcriptional initiation
Mediated through associations betwen various transcription factor activation domains and specific mediator subunit
Its role is consistent with the topological hierarchy (spacial organisation) observed in transcriptionally active looped-out chromatin
Mediates effect of enhancer elements and their binding proteins on the basal transcription machinery (RNA pol II)

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23
Q

Does transcription occur in a linear manner?

A

No. it is not, Mediator bridges sections of chromatins together to make enhancer closer

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24
Q

In prokaryotes, how does transcription occur differently than in eukaryotes?

A
  • prokaryotes lack membrane nucleus
  • Prokaryotes only have 1 RNA pol, not 3 different types
    1. When transcription starts, see a multiple RNA pol joining transcription start site and transcribing at the same time → transcription bursting
    2. Ribosomes bind to mRNA as it is being synthesized to start protein synthesis directly, doesn’t wait for mRNA to be completely synthesized
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25
Q

How can we visualize specific nascent transcript?

A
  • Not RNA-seq, PT-PCR or Northern bc they measure steady-state levels (consider the RNA synthesized - RNA degraded)
  • Add RNA structure close to start site
  • Use RNA binding protein fused to GFP which will be at the start of the transcripted RNA
    *GFP interacts with 5’ section of mRNA
26
Q

How does transcription occur in Eukaryotes (rhythm-wise)?
Which differs from prokaryotes

A

Occurs through successive bursts
In Eukaryotes, 1 transcript at the time with different frequencies for different genes

27
Q

What does transcriptional effiency correlates with?

A

transcriptional efficiency ~ burst frequency

Depends on the efficiency of the enhancer so highly transcribed genes have better enhancers and a higher burst frequency

28
Q

What is the process of dissolution and condensation of p-granules?

A

*p-granules are not actual cells/solids they are actually droplets

Dissolution: condensate → little droplets
Condensation: little inidvidual droplets → condensates (aggregate)

Happens along subunit diffusion, condensate as they go towards the tail

29
Q

Which domains are responsible for mediation of liquid-liquid phase separation?

A

Intrinsically disordered domains

30
Q

Which are the different element that can be found in liquid-liquid condensate of a transcription initiation?

A
  • DNA (enhancer segments, gene of interest)
  • RNA
  • RNA pol II
  • TF
  • acetyl-lysine
31
Q

Which elements are responsible for the valency of the transcription liquid-liquid condensate?

A
  • Electrostatic interactions
  • Post-translational modifications
  • Intrinsically disordered proteins
32
Q

What is the structure of the mediator?

A

Big multisubunit protein complex:
3 domains → head, middle, tail

Junction between the head and the middle domain is flexible which allows to take conformation to join different sites and interact with RNA pol II

In each domain, many subunit responsible for binding specific DNA binding proteins, not all are necessary for all transcriptions
All DNA bound activators interact with a single mediator complex

33
Q

Is the general mediator an element of the PIC?
(Pre-initiation complex)

A

NO, PIC = general transcription factors
Efficiency of the reaction is enhanced by the mediator complex
ChatGPT says yes

34
Q

What is the dynamic kissing model?

A

It is the fact that linearly distant part of DNA join in the mediator condensate to enhance and regulate transcription

35
Q

What causes the deconsentation of the transcriptional condensate?

A

When enough RNA is synthesized, electrostatic interactions cause dissociation of the condensate

RNA-dependent formation and dissolution of transcriptional condensate may account for the burst phenomenon

36
Q

What does transcriptional efficiency correlates with?
(In the Fluroescence intensity experiment (graphs) with sna shadow gene)

A

Transcriptional efficiency correlates with burst frequency
The increase in GFP fluorescence means starts of transcription = RNA synthesis, formation of a condensate
Decrease in GFP intensity = dissolution of the condensate

A little bit of RNA increases RNA pol II efficiency (increasing portion of the spike) until electrostatic interaction of RNA causes dissociation of condensate (decrease in the spike)

37
Q

What are the nucleosomes?

A

High-order structures of DNA bound strongly to Histone (H2, H3, H4) and wound up

Necessary structures that package the genomic DNA into the nucleus in the form of chromosomes

30-nm chromatin fiber of packed

38
Q

What is heterochromatin?

A
  • Condensed for of chromatin localized at nuclear envelope often near the nuclear pores
  • Considered transcriptionnally inactive
  • Genes within that region are silenced, can’t be accessed by Trancription Factors bc DNA too compact
  • For regions which transcription could be detrimental to the organism
39
Q

What is Euchromatin?

A
  • Delicate and thread-like
  • Abundant in actively transcribing cells
  • May represent DNA that i unwound to provide transcriptional template
  • RNA pol II and TF can access these regions
40
Q

Explain the Silent Mating Type Loci in Yeast chromosome III?

A
  • 3 genetic loci on chromosome III → genetically control the mating type of Saccharomyces cerevisiae
  • HMLalpha and MHRa loci are in silenced regions
  • Have to be haploid to have a mating type and mate
  • Recombination of mating sequence in MAT loci → can have 3 states, MATa, MATalpha MATa/alpha
  • If both sequences are moved, organism is diploid → can’t mate
41
Q

What does transcriptional repression depend on? (in the yeast silent mating type loci)

A

Transcriptional repression depends on recruitment of SIR proteins (silent information regulator)
*Silencers are cis-regulatory elements
- work outside of context of mating
- Can even block expression of tRNA genes (RNA pol III)

42
Q

What happens to genes expressed in telomeres?

A

They are silenced

43
Q

Which are the required factors for repression of the silent mating type loci?

A
  • RAP1=repressor activator protein 1 in yeasts
    Binds DNA in region of the silencer + binds to repetitive sequence in telomeres
  • SIR1: cooperates with RAP1, helps recruit SIR2, 3, 4
  • SIR2: histone deacetylase → more compact chromatin
  • , SIR3 and 4: structural proteins that bind to hypoacetylated histone tails (H3, H4)
    *SIR = Silent Information Regulator in yeasts
44
Q

What did genetic screens identified?
(related to matin type loci)
What specifically is seen in the in situ hybridization/immunofluorescence?

A

It identified factors required for repression of the silent mating type loci

Telomeres and SIR3 (Silent Informational Regulator 3) pictured locations overlap as SIR3 keeps telomeres in heterochromatin form (probably by RAP1 recognition)

45
Q

What are some general post-translational modifications on histones?

A
  • Phosphorylation
  • Methylation
  • Acetylation
  • Ubiquitination
    *Electrostatic interactions between histone tails and phosphate backbone of DNA dictates chromatin state
    *Specific modifications on tails of H3 and H4 induce changes in chromatin structure
46
Q

Name 3 specific post-translational modifications on histones? (histone marks)

A

Transcriptional activator: H3K4me3 (3rd Histone, 4th Lysine, trimethylation)
Transcriptional inactivator: H3K9me3
Transcriptional inactivator: H3K27me3

47
Q

What is an epigenic traits/epigenetic marks?
Give are 3 example.

A
  • They are heritable traits (following cell division) transmitted independently of the DNA sequence itself
  • DNA marks (methylations) are read by specific proteins then used to modify histones in proximity through mSin3 recruitment (mammalian Sin3 comes in and changes histones around the region)
48
Q

What reaction is responsible for epigenetic DNA marks?

A

Methylation
Read by specific proteins then used to modify histones in proximity through mSin3 recruitment

49
Q

Give 3 examples of epigenetic traits/marks?

A
  • Inactive X (Xist, histone methylation and heterochromatin spreading, in female mammals)
  • Developmental restrictions → Polycomb = group of proteins making sure some regions of specific cells are never expressed (so humans don’t have antennae)
  • Imprints (DNA methylations)
  • Histones deacetylations
50
Q

What is the role of mSin3?

A

mammalian Switch-Insensitive 3 = protein playing regulatory role in gene expression
Sin3 complex associated with removal of acetyl groups from histone proteins leading to more compact chromatin structure (repressive effect on transcription)

51
Q

What are epigenetic readers and writers?

A

Epigenetic readers: Ensuring every cell daughter acquires the appropriate epigenetic profile, proteins that recognize the marks

Epigenetic writers: protein complex that put the marks down, heritable following cell division

52
Q

What post-translational modification is required for maintenance of heterochromatin during DNA replication?
(How does it happen)

A

H3K9me3

Following replication, only half of histones are trimethylated so histone methyltransferase (HMT) recognizes H3K9me3 and know the naive neighbouring histones should also be trimethylated so methylates them
H3K9me3 acts as initiation point/nucleation point for HMT

HMT(Histone Methyltransferase) therefore acts as a epigenetic reader and writer

53
Q

How can antibodies be used to identified regions of the genome affected by histone modifications?

A

Antibodies for specific reactions on specific residues + ChiP (Chromatin Immunoprecipitation)
ex: Antibody against H3K9me3

  1. Using know primer of you want to know whether a specific gene is affected
  2. Using NGS, can analyze the entire genome to know what regions of the genome are affected
54
Q

How do transcriptional activators work?

A

Transcriptional activators recruit Histone Acetyl Transferases (HATs)

Acethylation (adding acetyl groupsto lysine residues on histones) neutralizes positive charge on lysine residues → neutralizes the electrostatic interaction between Histone and DNA → complex formation

Some transcriptional activators can overcome the repressed chromatin state by inducing acetylation of histone tails through associated proteins

They promote formation of Euchromatin

55
Q

How do histones interact with DNA?

A

Positively charged lysines and arginines on N-terminal histone tail interacts electrostatically with the DNA phosphate groups

56
Q

How does Activator-directed histone hyperacetylation work in yeasts?

A
  • Gcn4 binds to UAS (yeast enhancer)
  • Has its Co-activator Gcn5 which interacts with neighbouring histones → Hyperacetylation of histone N-terminal tails (adds more acetyl groups to histone tails)
  • Addition of acethyl groups releases the compactness of histones
57
Q

How to transcriptional repressors work?
Give an example.

A

Transcriptional repressors act through histone deacetylation complexes (HDACs)

Example:

Rpd3p (in yeast) = HDAC → removes acetyl groups → more positive histone tails → + interaction with DNA backbone → compaction

Ume3p (yeast TF) targets and binds URA with Sin3 and co-repressor Rdp3p which removes acetyl groups from histone tails
*Sin3 = scaffold protein which binds TF and HDAC

58
Q

How does LacI interact with DNA chromatin?

A

LacI bacterial transcriptional repressor, forms a tightly compact puncta where recongnizes DNA binding sites → enhances formation of heterochromatin

59
Q

What is VP16 AD?
What happens when it is bound to the LacI promotor?

A

A strong activator (decondenses chromatin into euchromatin to allow transcription)

Bound with LacI promotor, can decondense heterochromatin regions that would normally stay silent (regions that are normally bound to by Lac)

*ATP-dependent

60
Q

What are chromatin remodellers?

A

Protein/protein complexes responsible for changes in chromatin conformation/DNA accessibility for transcription
ATP-dependent activity

ex:
SWI/SNF (Switch/Sucrose Non-Fermentable)
LacI-VP16 AD

61
Q

What are Pioneer transcription factors

A

DNA binding transcriptional activators/repressors that interact with DNA sequences exposed on outside of the DNA-wound histone octamer
Work with co-activators

Recruit enzymes that alter confirguration of the neighbour histone tails → opens chromatin to permi association with general TF

Amoung first to activate genes during embryogenesis

62
Q

How can chromatin be seen for transcriptional activation?

A

It is a barrier for transcriptional activation

63
Q

What are the elements of a co-repressor or co-activator complex?

A

DNA binding transcription factor + Pioneer factors

BIOL 200 (11 decks)

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