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Molekularbiologie > Regulation of gene expression > Flashcards

Regulation of gene expression Flashcards

Basal & regulated transcription

1
Q

Which transcription factor mutation causes
anomaly of the development of the limbs („synpolydactyly”)?

A

HOXD13

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2
Q

Which direction is the template stand being read?

A

3’ to 5’

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3
Q

Which direction is RNA being transcribed?

A

5’ to 3’

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4
Q

3 main steps of transcription

A

Initiation, elongation, termination

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5
Q

Elements involved in RNA transcription

A
  1. DNA (template)
  2. Transcription unit: from promoter to terminator
  3. DNA elements: Core promoter, enhancers, silencers
  4. RNAP II holoenzyme: protein complex recruited to the promoter = RNAP II + other factors
  5. Transcription factors: general TFs and regulatory TFs
  6. Transcription apparatus = RNAP II holoenzyme + regulatory TFs
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6
Q

Methods for the elucidation of RNA transcription

A

EMSA, footprints, protein purification and biochemistry, in vitro transcription
assays, IP and chromatin IP

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7
Q

Methods for the elucidation of RNAP structure

A

Single particle cryoEM

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8
Q

What is Single particle cryoEM and how does it work?

A

Method to determine high resolution structure
Purified sample -> Sample onto grid -> Freeze -> cryoTEM -> Data acquisition -> 2D projections

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9
Q

What is DNase I footprinting?

A

Method to identify protein-DNA interactions; DNA sequences bound by proteins are protected from enzymatic digestion by DNase I

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10
Q

What is EMSA = Electrophoretic Mobility Shift Assay?

A

Method to identify protein-DNA(/RNA) interactions; DNA-Protein complexes migrate more slower than unbound DNA(/RNA)

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11
Q

What is Chromatin Immunoprecipitation - ChIP?

A

Method to study protein-DNA interactions - where specific proteins, such as transcription factors, histones, or other chromatin-associated proteins, bind on the genome
Crosslinking -> Cell lysis -> DNA fragmentation -> Immunoprecipitation -> Reverse crosslinking and DNA purification -> Detection or sequencing

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12
Q

Types of polymerases

A
  1. DNA-dependent DNA polymerases (DNA → DNA) - Enzyme of DNA replication
  2. DNA-dependent RNA polymerases (DNA → RNA) - Enzyme of RNA transcription
  3. RNA-dependent RNA polymerases (RNA → RNA) - Viral polymerases
  4. RNA-dependent DNA polymerases (RNA → DNA) - Viral RT, endogenous RT (retroposons), telomerase
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13
Q

What is RNA Polymerase I responsible for and where is it located? How does it react to α-Amanitin?

A

pre-rRNA (for 28S,18S, 5,8S rRNA)
In the nucleolus, resistant to α-Amanitin inhibition

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14
Q

What is RNA Polymerase II responsible for and where is it located? How does it react to α-Amanitin?

A

pre-mRNA; 4 snRNA; many pre-miRNAs, lncRNAs
In the nucleoplasm; inhibited by α-Amanitin

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15
Q

What is RNA Polymerase III responsible for and where is it located? How does it react to α-Amanitin?

A

pre-tRNA, 5S rRNA; snRNAs; some pre-miRNAs
In the nucleoplasm; partially inhibited by α-Amanitin

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16
Q

RNAP II Structure

A
  • Core domain: horseshoe-shaped, catalytic
    activity in its centre;10 subunits (RPB1, RPB2
    and other smaller subunits)
  • Stalk domain: protrudes out of core, mobile structural element that functions primarily in transcription initiation and elongation, especially under stress conditions; 2 subunits (RPB4, RPB7 for interactions with TFs and stabilisation)
  • ~ 500 kDa und 12 Untereinheiten
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17
Q

Distinct functions of different subunits and domains of RNAP II

A

*Binding of DNA
*Binding of the DNA-RNA hybrid
*Polymerisation of the RNA
*„proofreading “
*Progression on the template
*Directing the RNA product to the exit

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18
Q

Where is the clamp domain of RNAP II found? Whedn does it open and closed?

A

Clamp domain is in RBP1
Open: during RNA insertion & after termination to release the enzyme.
Closed: during elongation

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19
Q

Where is C-terminal domain (CTD) of RNAP II found?

A

C-terminal domain (CTD) is in RPB1; it is unstructured

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20
Q

What are General / basal TFs (GTFs)?

A

Small subset of TFs necessary for the
initiation of transcription. They are part of the PIC (pre-initiation complex = RNAP II + GTFs on the promoter) and are required for
initiation by RNAP II at all promoters. Expressed at high and stable levels.
Examples: TFIIB, TFIIH

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21
Q

Regulatory / Specific TFs

A

Regulate gene expression (activators /
repressors). Large subset of TFs. Low abundance, variable expression.
Examples: NF-κB, AP-1

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22
Q

RNAP + GTFs = ?

A

RNAP holoenzyme

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23
Q

What is the rate of basal transcription?

A

low

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24
Q

How is transcription regulated?

A
  • „Enhancers“ and „silencers“ are short DNA sequences that serve as binding sites
    for activating / repressing proteins which interact directly or indirectly with RNAP II.
  • proximal, distal, upstream, downstream
  • Their function involves secondary structures of the DNA, which are
    stabilized by the cohesin/CTCF complex
  • Insulators are additional regulatory elements.
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25
Q

Core promotor

A

Shortest DNA sequence required by RNAP II to initiate transcription

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26
Q

What is responsible for recognizing the promotor DNA sequence?

A

GTFs, not RNAP

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27
Q

What are frequent core promotor sequences?

A
  • no universal core promoter
  • ~ 50% of the core promoters have a TATA box (~-25 to -35 bp upstream of TSS)
  • „TATA-less“ promoters often contain a DPE (downstream promoter element) (+28 to +32 bp)
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28
Q

Focused promotors

A

Promoters with TATA box; transcription starts from a narrowly defined site.
Often on cell type- specific genes

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29
Q

Broad promotors

A

Hypomethylated CpG islands; able to initiate transcription from multiple sites (in both directions!).
Often in housekeeping genes

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30
Q

Which elements come in direct contact with DNA?

A

TFIID, TBP and TFIIB

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31
Q

What must happen to the chromatin before transcription initiation?

A

Before transcription initiation can begin, the chromatin has to be modified and remodeled to the open configuration, and nucleosomes have to be moved or removed from the promoter region!
-> Then only can the RNAP positioning factor bind the DNA

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32
Q

What does TFIIB bind to?

A

BRE = TFIIB Recognition Element (upstream and downstream) (C/G)

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33
Q

What does TBP bind to?

A

TATA
* TBP = TATA-Binding Protein; only in the case of RNAP II (and in rare cases for RNAP III) that TBP binds the TATA box in the core promoter/different role for RNAP I

34
Q

What does TFIID bind to?

A

INR = Initiator (C/T) & DPE = Downstream Promoter Element & MTE = Motive Ten Element

35
Q

How many subunits does TFIIA have and what does it interact with?

A

3, TBP

36
Q

What is the function of TFIIA?

A

Stabilization of TBP binding to DNA & PIC complex
* TBP = TATA-Binding Protein; only in the case of RNAP II (and in rare cases for RNAP III) that TBP binds the TATA box in the core promoter/different role for RNAP I; universal factor of the positioning complex

37
Q

How many subunits does TFIIB have and what does it interact with?

A

1(several structural domains: ribbon, linker, reader, core - cyclin domains), TBP and acidic activators

38
Q

What is the function of TFIIB?

A

Recruitment of RNAPII and involvment in selection of site of initiation

39
Q

How many subunits does TFIID have and what does it interact with?

A

≥ 5 (=TBP + TAFs), DNA(TATA Box, Inr, DPE, MTE) and proteins (TFIIA, TFIIB, TFIIE, RNAP II, activators, chromatin modifiers, among others)

40
Q

What is the function of TFIID?

A

Positioning of RNAPII complex and recognition of the core promoter to position the RNAP; nucleation of transcription; specific functions of TAFs (TAF250: HAT, UbQligase)
* TFIID is a protein complex (~750 kDa)
* TFIID is the largest GTF
* TFIID = TBP + several TAFs (TBP-Associated Factors)

41
Q

How many subunits does TFIIE?

A

2

42
Q

What is the function of TFIIE?

A

Modulatition of acitivity of TFIIH and role in promoter escape

43
Q

How many subunits does TFIIF have and what does it interact with?

A

2, RNAP II and TFIIB

44
Q

What is the function of TFIIF?

A

Promotion of stable association of RNAP II
with TFIIF-TBIIB-core promoter complex

45
Q

How many subunits does TFIIH have and what does it interact with?

A

~10(kinase: CDK7, 2 helicases:XBP + XPD), kinase complex (TFIIK)

46
Q

What is the function of TFIIH?

A

Activation of RNAP II, promoter melting, phosphorylation of CTD
CDK7 and cyclin H: kinase activity
ERCC2/XPD & ERCC3/XPB: helicase & ATP-ase activity, also involved in NER (nucleotide excision repair)

47
Q

How many proteins does RNAP II PIC contain?

A

More than 70 proteins

48
Q

What is the first step in the assembly of the PIC?

A
  • TBP interacts with TAFs
  • TFIID binds and bends the DNA by 80 – 90 degrees.
  • Drastic conformational changes of TFIID
  • Further general TFs enter the complex,
    including TFIIA and TFIIB
49
Q

What is the second step in the assembly of the PIC?

A
  • TFIIB establishes several protein-protein
    interactions with RNAP II
  • B-ribbon: stabilizes the RNAP II complex at the TSS region (recruitment)
  • C-terminal Cyclin Domain: Binding to TBP and upstream DNA
50
Q

What is the third step in the assembly of the PIC?

A
  • Recruitment of RNAPII and the TFIIF
  • PIC core is complete!
  • Subsequent binding of TFIIE and TFIIH completes the inactive, closed form of the PIC
  • PIC forms a channel enclosing the template DNA over the TSS region
51
Q

Which GTF is responsible for the recruitment of RNAP II?

A

TFIIB (B-ribbon)
* Inserts into the RNA exit channel of RNAP II
* Other TFIIB domains also interact with the RNAP II catalytic domain
* This facilitates the initiation of transcription but leads to a steric hindrance / steric clash when the RNA grows

52
Q

Which GTF is responsible for the activation of RNAP II within PIC?

A

TFIIH

53
Q

What is XBP?

A

XBP is a subunit of TFIIH
3’ to 5’ ATP-dependant helicase

54
Q

What is XPD?

A

XPD(=Ssl2) is a subunit of TFIIH
5’ to 3’ ATP-dependant helicase

55
Q

How does the activation of RNAP II within PIC occur?

A
  • TFIIH helicase XPB helps unwinding the DNA duplex at the TSS (this requires ATP)
  • Formation of the transcription bubble
  • Conversion of the inactive, closed PIC into the active, open PIC.
  • Transcription starts

(But transcription can still pause or abort)

56
Q

What is mainly required for successful elongation?

A

Sequential phosphorylation of several proteins
e.g. the RNAP CTD ist phosphorylated by the TFIIH CDK7

57
Q

What does promotor escape depend on?

A
  1. TFIIH unwinds DNA and phosphorylates the CTD
  2. RNA-DNA Hybrid stability ensures a stable foundation for RNAP II to move downstream.
  3. CTD Phosphorylation marks the transition to elongation and releases RNAP II from the promoter.
  4. ATP Hydrolysis provides energy for the structural transitions required in RNAP II

→ promoter escape depends on a competition between TFIIB and the transcript (RNA)

58
Q

How does promotor escape occur?

A
  • CTD phosphorylation (Ser5) by CDK7 (TFIIH) (release of the mediator) -> Initiation of a short capped transcript
  • Release of General Transcription Factors; TFIIB, TFIIE, and TFIIH
  • RNAP II moves downstream, leaving the core promoter and transitioning into elongation
59
Q

How does productive elongation occur?

A
  • P-TEFb/CDK-cyclin T phosphorylates the RNAP II CTD at Ser2 but also DSIF und NELF
  • Phosphorylated NELF dissociates from the complex
  • Phosphorylated DSIF converts to a positive elongation factor (holds RNAP II clamp in „closed“ conformation) → productive elongation
60
Q

What causes promotor-proximal pausing?

A

Negative elongation factors NELF and
DSIF prevent elongation

61
Q

What causes productive elongation?

A

Recruitment of further elongation factors -> during elongation, Thr4 becomes phosphorylated

62
Q

Methods of elucidation of the PIC assembly

A

EMSA & Footprint

(Association of RNAP II on the core promoter with the TFIID-TFIIB-TFIIA complex depends on TFIIF)

63
Q

What happens after the first 5-6 ribonucleotides are assembled at the initiation of transcription?

A

Steric hindrance which results in competition between RNA elongation and interaction with TFIIB
2 possibilities:
1. TFIIB „wins“→ abortive transcription → new attempt
* RNA „wins“ → TFIIB, TFIIE, & TFIIH are released → „promoter escape“→ Elongation

64
Q

Where is CTD (C-terminal
domain) found?

A

RPB1 which is the largest subunit of RNAP II
It is unstructured

65
Q

How long are the CTD-repeats in mammals?

A

52 repeats
First (N-terminal) 26 repeats are well conserved and essential
Last (C-terminal) 26 repeats show some variability.

66
Q

How long are the CTD-repeats in yeast?

A

26 repeats

67
Q

How is the activitiy of RNAP II regulated?

A

Covalent modifications of
its CTD, e.g. by TFIIH/CDK7, P-TEFb/CDK9 and CDK8 kinases.

68
Q

What is the CTD of RNAP II responsible for?

A
  • Initiation of DNA transcription
  • Capping of the RNA transcript
  • Attachment to the spliceosome for RNA splicing
69
Q

What regulates RNAP II?

A
  • Post-translational modifications of CTD
  • Further processes associated with RNA transcription (e.g. mRNA processing and export)
70
Q

What is the consensus repeat of the CTD of RNAP II?

A

YSPTSPS

71
Q

When does phosphorylation of the CTD occur during transcription?

A
  1. Recruitment of RNAP II to the promoter
  2. Promoter-proximal pausing (within ~100 nucleotides)
  3. Productive elongation
  4. Termination
72
Q

Wich amino acids are phosphorylated during transcription?

A

Ser5, Ser7, Tyr1, Ser2, Thr4

73
Q

What does the phosphorylation of Ser 5 residues by CDK7 of TFIIH cause?

A
  • Reduces the affinity of RNAP II for promoter-associated transcription factors -> promotr escape
  • Facilitates the recruitment of RNA capping enzymes
74
Q

What does the phosphorylation of Ser 2 residues by pTEFb in the CTD cause?

A

Promotes productive elongation by recruiting elongation factors and RNA-processing enzymes

75
Q

What does the phosphorylation of Thr 4 residues by CDK12/13 in the CTD cause?

A
  • Facilitates productive elongation by stabilizing interactions with elongation and RNA processing factors
  • Involved in histone modification coupling
  • 3’-end RNA processing
76
Q

What happens during termination of transcription?

A
  • Combination or Ser2 and Thr4 phosphorylation serves as signal for transcription termination, RNA cleavage and polyadenylation.
  • Recruitment of termination factors and RNA
    processing factors
  • Initial phosphorylation marks are
    consequently partially or completely
    removed by specific phosphatases
  • RNAP II recycling.
77
Q

Which mutations cause Xeroderma pigmentosum?

A

Mutations in helicases XBP & XDP of TFIIH

78
Q

What does CDK9 of P-TEFb do?

A

Phosphorylates Ser2 for elongation

78
Q

What does CDK7 of TFIIH do?

A

Phosphorylates Ser5 during transcription initiation

79
Q

What do CDK12/CDK13 do?

A

Phosphorylate Thr4 for productive elongation and RNA processing

Molekularbiologie (9 decks)

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