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BMD211-Human Molecular Biology > Lecture 7: Mechanisms of Eukaryotic Transcription > Flashcards

Lecture 7: Mechanisms of Eukaryotic Transcription Flashcards

- What is transcription? - RNA polymerases - Mechanism of basal transcription

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1
Q

What is transcription?

A
  • Transcription is 5′ to 3′ on a template that is 3′ to 5′
  • Coding (nontemplate) strand – The DNA strand that has the same sequence as the mRNA and is related by the genetic code to the protein sequence that it represents
  • RNA polymerase: An enzyme that synthesizes RNA using a DNA template
  • > formally described as a DNA-dependent RNA polymerase
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2
Q

Describe and explain the structure of a gene

A
  • Promoter: A region of DNA where RNAP binds to initiate transcription
  • Starpoint: The position on DNA corresponding to the first base incorporated into RNA (begins at +1)
  • Terminator: A sequence of DNA that causes RNAP to terminate transcription
  • Transcription unit: The sequence between sites of initiation and termination by RNAP; it may include more than one gene
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3
Q

Explain RNA polymerisation

A

The 3’-OH group in ribose of the last ribonucleotide reacts with an incoming ribonucleoside 5’ triphosphate-> to be added to chain

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4
Q

State how many subunits does each RNAP have

A 
I= 14
II= 12
III= 17
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5
Q

State where each RNAP is located and their RNA products

A

I-> nucleolus-> pre-rRNA
II-> nuceloplasm-> pre-mRNA= some snRNAs
III-> nuceloplasm-> t-RNA, 5s rRNA, U6 snRNA + 7SL RNA

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6
Q

List the RNA subunits that are shared

A

Rpb5, Rpb6, Rpb8, Rpb10, Rpb12

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7
Q

Explain what the transcription bubble is

A
  • RNAP separates the 2 strands of DNA in a transient “bubble.”
  • Uses 1 strand as a template to direct synthesis of a complementary sequence of RNA
  • The length of the bubble is ∼12 to 14 bp
  • The length of RNA-DNA hybrid within it is ∼8 to 9 bp
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8
Q

Explain the overview of transcription

A
  • RNAP binds to a promoter site on DNA to form a closed complex
  • RNAP initiates transcription after opening the DNA duplex to form a transcription bubble =the open complex
  • RNAP the escapes the promoter from the initiation site
  • Elongation-> transcription bubble moves along DNA
  • The RNA chain is extended in the 5′–3′ direction by adding nucleotides to the 3’ end
  • When transcription terminates or stops-> the DNA duplex reforms + RNAP dissociates at a terminator site
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9
Q

Explain the importance of TFs

A
  • RNAP cannot initiate transcription from specific start sites without the assistance of other proteins-> transcription factors
  • Each RNAP has its own specific set of transcription factors (TFIX, TFIIX, TFIIIX)
  • > assist in locating Pol I, II or III transcriptional start sites
  • Around each transcription start site is a core promoter
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10
Q

Explain what focused initiation is

A
  • > starts from a single nucleotide or within a cluster of several nucleotides
  • > Predominant means of transcription in simpler organisms
  • > Regulated genes tend to have focused promoters
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11
Q

Explain what dispersed initiation is

A
  • > there are several weak transcription start sites over a broad region of about 50 to 100 nucleotides
  • > observed in approximately two-thirds of vertebrate genes
  • > constitutive genes typically have dispersed promoters
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12
Q

Describe and explain the Eukaryotic core Pol II promoter motifs

A
  • TATA box: TATAXAX consensus sequence (X is A or T) located 25-30 bp upstream of the transcription start site
  • Initiator element (Inr): overlaps the transcription initiation site
  • Downstream promoter element (DPE): extends from about +28 to +34
  • TFIIB recognition element (BRE)
  • XCPE1: present in TATA-less human core promoters (1% human genes)
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13
Q

Explain the transcription initiation process

A
  1. TBP region of TFIID recognises TATA box with aid of TFIIA
  2. TFIID + TFIIA recruit TFIIB
  3. TFIIB binds to complex
  4. TBP bends the TATA box around the C terminal domain of TFIIB
  5. The N-terminal domain of TFIIB brings the complex to RNAPII to join+ positions the transcription initiation site in the active site of RNAP
  6. The RNAPII-TFIIB- promoter complex recruits TFIIE and binds
  7. TFIIE recruits TFIIH and bind-> PIC formed + closed complex
  8. Helicase activity of TFIIH unwinds DNA in the area of the initiation site
  9. TFIIF captures the non-template strand after unwinding
  10. The template strand descends to the active site-> open complex
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14
Q

Explain the function of each TFIID in the PIC

A

-TBP is the TATA binding subunit of TFIID
Binds TATA element+ deforms promotor
-Platform for the assembly of TFIIB + TAFs
-Bind Inr, MTE, DPE + DCE promotor elements

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15
Q

Explain the function of each TFIIA in the PIC

A

-Stabilises TBP +TFIID binding –Blocks inhibitory effects of TAF1 + other proteins

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16
Q

Explain the function of each TFIIB in the PIC

A
  • Stabilises TFIID- promotor binding
  • Contributes to transcription start site selection
  • Helps to recruit RNAPII-TFIIF to core promotor
17
Q

Explain the function of each TFIIF in the PIC

A
  • Binds RNAPII
  • involved in recruiting polymerase for PIC
  • Required to recruit EFIIE+EFIIH to PIC
18
Q

Explain the function of each TFIIE in the PIC

A
  • Helps to recruit TFIIH to core promotor

- required for promotor melting

19
Q

Explain the function of each TFIIH in the PIC

A
  • Functions in transcription + DNA repair
  • has kinase+ helicase activities
  • is essential for open complex formation
20
Q

Explain what TBP is and what it does

A
  • TATA-binding protein (TBP) is saddle shaped and bends DNA by 80°
  • Interaction between TBP and the TATA box involves conformational changes in both TBP and the TATA box
  • TBP interacts with TATA in the minor groove
21
Q

Explain what TAFs are and what they do

A
  • TBP-associated factors (TAFs) bind promoter elements other than TATA
  • Additional proteins present in TFIID are called TBP-associated factors (TAFs)
  • TFIID contains a core set of 13 TAFs
  • TAFs are required for high levels of transcription & to transcribe genes that lack a TATA box
  • TATA-less promoters ->the TAFs that bind to regulatory elements
22
Q

Explain the overview of transition from initiation to elongation

A
  1. Open complex formation: initiation of transcriptional bubble
  2. RNA chain initiation: first 2 ribonucleotides line up on template strand in the transcription bubble + RNAP catalyses phosphodiester bond formation
  3. Abortive transcription: formation of short transcripts which are released
  4. Promoter escape: chain extension beyond 7 ribonucleotides-> triggers TFIIB release
    =formation of the transcriptional elongation complex
23
Q

State what part of RNAP is phosphorylation for elongation

A

Carboxyl terminal domain (CTD) of the largest subunit in RNAP 2 (Rpb1)

24
Q

List the properties of TFIIH

A
  • 10 subunits
  • ring shaped
  • 5’3’ and 3’ 5’ helicase activities
  • cyclin-dependent protein kinase (CDK) activities
  • ATPase
25
Q

Explain the phosphorylation process of CTD for promoter clearance

A
  1. Phosphorylation of Ser5 by TFIIH-> recruits 5’ end capping enzymes on primary transcript
  2. Phosphorylation of Ser 2 by P-TEFb-> activates elongation, splicing + poly adenylation
  3. Phosphorylation on Ser7 by unknown kinase-> ?
  4. Dephosphorylation of Ser 5 by Ser5 phosphatase
  5. Polyadenylation factors bind to Ser2 phosphorylated repeats w/ peptidyl-prolyl bonds in trans configuration
  6. Dephosphorylation of Ser2 by Ser2 phosphatase
  7. Dephosphorylation of Ser7 by potential Ser7 phosphatase
  8. RNAPII able to be recycled + bind to another promotor
26
Q

Explain the importance of phosphorylation of CTD

A
  • Phosphorylation of CTD is involved in processing mRNA transcript
  • > provides binding/recognition sites for mRNA processing
27
Q

Describe the overview of elongation

A
  • More stable than the initiation complex
  • Approx. 14 bp are melted to form the transcription bubble
  • First eight nucleotides within bubble are paired with the RNA chain
  • Double stranded DNA opens up in front of the bubble and closes up behind the bubble as RNAP moves along
28
Q

Explain why RNAP doesn’t have a steady pace during elongation

A
  • elongation is temporarily delayed at pause sites
  • pausing -> arrest and termination
  • > arrested by Mg ion at active sit or RNAPII
  • > arrested is an important step in proofreading
  • TFIIS role -> reactivates arrested RNA polymerase II
29
Q

Describe what makes up the promoter for RNAP I

A
  • RNA polymerase I promoters consist of:
  • core promoter
  • upstream promoter element (UPE)
30
Q

Explain how RNAP I binds to the promoter

A
  • The factor UBF1 binds to UPE -> wraps DNA around a protein structure
    = brings core and UPE close together
  • RNAP I holoenzyme= core binding factor SL1 + RNAP I
    ->binds to core promotor
  • SL1 includes TBP factor-> involved in initiation by all 3 RNAPs
  • RNAP I binds to the UBF1-SL1 complex at the core promoter
31
Q

Describe what makes up the promoter for RNAP III

A
  • RNAP III has 3 types of promoter- 2 of which are internal promoters
  • Internal promoters: have short consensus sequences located within the transcription unit
  • > cause initiation to occur at a fixed distance upstream
  • Upstream promoters: contain three short consensus sequences upstream of the start point that are bound by transcription factors
32
Q

Explain how RNAP III binds to the promoter

A
  • TFIIIA + TFIIIC bind to the consensus sequences

= enable TFIIIB to bind at the startpoint
- TFIIIB has TBP as one subunit -> enables RNA polymerase to bind
= PIC formed

33
Q

state the difference between Internal type 1 pol III promoter +Internal type 2 pol III promoter

A
  • Internal type 2 pol III promotor-> 2 TFIIIC bind to short consensus sequences
  • Internal type 1 pol III promotor -> TFIIIA + TFIIIC bind to short consensus sequences

BMD211-Human Molecular Biology (5 decks)

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