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BIOC0020 > L1 : Pol III and Gene Regulation > Flashcards

L1 : Pol III and Gene Regulation Flashcards

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1
Q

What is gene expression?

A

Process by which a DNA gene produces a functional product
Usually protein but also RNA (eg. miRNAs, lncRNAs)
Genome encodes many proteins, which define cell function

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2
Q

Different ways to control gene expression?

A

Controlling gene expression controls cell function
Major point of control is regulation of transcription

Other ways:
- RNA export from nucleus
- Stability (inside/outside nucleus)
- Splicing
- Translational control

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3
Q

Draw a diagram showing different points of gene expression regulation

A
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4
Q

Describe features of nucleus

A

Nucleus is not uniform (never naked DNA within cells)
All eukaryotes tightly package their DNA genome with histone proteins into chromatin complex to fit into nucleus
Interactions between histones and DNA are electrostatic (negative backbone phosphates on DNA)

Nucleolus - rRNA transcription, ribosome biogenesis

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5
Q

Compare euchromatin and heterochromatin?

A

Heterochromatin = densely packed and highly condensed
- Transcriptionally inactive (silenced)

Euchromatin = loosely packed and less condensed
- Transcriptionally active (expressed)

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6
Q

How do chromatin modifications allow events on DNA?

A

Condensed chromatin is repressive
How to relax/open it?
- Histone variants
- Chromatin remodellers
- PTMs on histone tails

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7
Q

What comprises each RNA polymerase and what do they transcribe?

A

RNA Pol 1 - rRNA
(14 subunits)
RNA Pol 2 - mRNA
(12 subunits)
RNA Pol 3 - tRNA
(17 subunits)

Pol 1 and 3 transcription can represent up to 80% all transcripts

Coding strand = non-template strand
Note: prokaryotes have 1 RNAP

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8
Q

Why do RNAPs have different numbers of subunits?

A

Specialised for different transcriptional roles
- Varied complexity of targeting and regulation

Some subunits and GTFs are specific for each system (eg. Rpb1 (1), A190 (2), C160 (3)) and some are shared

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9
Q

What is required for transcription at every gene (Pol 1/2/3)?

A

At every gene (Pol 1/2/3)
- RNA polymerase
- NTP substrate
- DNA template (separated)
- General transcription factors (GTFs)
Note: highly purified RNA pol + NTP + template - no polymerisation (need GTFs)

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10
Q

What is required for transcription at mRNA genes (Pol 2)?

A

At mRNA genes (Pol 2)
- Some genes require activators (TFs)
- Co-activators (chromatin modifying enzymes and remodellers)

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11
Q

What is the pre-initiation complex?

A

Large multi-protein assembly that forms at the promoter region of a gene during the early stages of transcription

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12
Q

What is the role of the PIC?

A

Positions RNA pol correctly and facilitates unwinding of DNA

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13
Q

What is the anatomy of an mRNA gene?

A

Core promoter, TSS, 5’ UTR, start codon, ORF, stop codon, 3’ UTR, termination

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14
Q

What is the role of the core promoter?

A

Core promoter contains sequence elements that GTFs can recognise in order to start transcription

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15
Q

What are key elements of the promoter?

A

TATA-box - TATA binding protein flanked by:
- BreU - TFIIB recognition element upstream
- BreD - TFIIB recognition element downstream

Initiator - marks the transcriptional start site

DPE - downstream promoter element

MTE - motif ten element

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16
Q

What are the requirements to begin mRNA transcription (pol 2) successfully?

A

Core promoter DNA
RNA Pol 2 recruitment and positioning
Strand separation/melting
TSS selection

Note: highly purified Pol 2 inactive on double strand

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17
Q

How do GTFs (basal) provide these activities?

Study These Flashcards
A

GTFs absolutely required for Pol2 activity: TFIIA, B, D, E, F, H
Most are protein complexes
Some bind core promoter elements directly
Some bind other GTFs and RNAP

When assembled, complex of RNAP2 and GTFs is PIC

18
Q

What mnemonic helps remember order for PIC formation?

Study These Flashcards
A

Detect
Anchor
Bridge
Facilitate
Recruitment
Expose
Helicase

19
Q

What is the process of formation of PIC?

Study These Flashcards
A
  1. Recognition of core promoter by TBP-TFIID and interactions form between TBP and TATA box
    (TFIID can also recognise INR and DPE elements and act as coactivator
  2. When TBP bound, TFIIA stabilises interaction between TBP and DNA
  3. Recruitment of TFIIB that assists in stabilisation, also recognising BreU and BreD elements
    (Assists with promoter opening and TSS selection)
  4. TFIIF assists in binding of RNAP2 to promoter bound TBP-TFIIB complex
    (Also provides platform for later GTFs to bind)
  5. TFIIE assists in promoter opening and recruitment of TFIIH
  6. TFIIH contains ATP driven helicase enzyme
    (Translocates along DNA and works to open promoter to present template strand for base pairing with NTPs)
20
Q

What does pol 3 transcribe?

Study These Flashcards
A

Short (>350 nt) and untranslated RNAs (tRNAs, 5S rRNA, U6 snRNA)
House keeping genes

21
Q

What are the unique features of Pol 3 transcription?

Study These Flashcards
A
  • Extreme stability of TF3B
  • No ATP hydrolysis required
  • Termination at polyT
  • Facilitated reinitiation
  • Direct redox regulation by Brf2
22
Q

What are the 3 promoter types of Pol 3?

Study These Flashcards
A

Type refers to how and where required TFs arranged in relation to gene

Type 1:
- eg. 5S rRNA
- Internal control elements
Type 2:
- eg. tRNA
- Internal control elements
Type 3:
- Vertebrates only
- eg. U6 snRNA
- Upstream promoter
- SNAPc involved in recruitment

23
Q

What differs between Pol 3 promoter types?

Study These Flashcards
A

Requires different PICs and recruits different combination of TFs

24
Q

What is TFIIIB composition depending on promoter type?

Study These Flashcards
A

Type 1 + 2: Bdp1, Brf1, TBP
Type 3: Bdp1, Brf2, TBP

Brf1 used with internal promoters, part of TFIIIC-dependent pathway
Brf2 used with upstream promoter, TFIIIC-independent, instead recruited by SNAPc

25
Describe the structure of Brf1 and Brf2
Components of TFIIIB Similar architecture with 2 cyclin repeats for DNA interaction Brf2 possesses unqiue long C-terminal extension, also associated with redox regulation
26
What is the role of the molecular pin in redox-sensitive DNA binding of Brf2?
1. Molecular pin has conserved Cys that makes non-covalent interactions with DNA 2. Oxidising cysteine increases size and gives charge 3. No longer fits within cavity as electrostatically repulsed (-ve phosphate backbone) 4. Assembly of complex is inhibited by presence of oxidised Cys
27
How was EMSA used to study redox-sensitive DNA-Brf2 binding?
Electrophoretic mobility shift assay - Mutate Cys in brf2 to aspartate - Mimic cys ox (sulfinic), introdycing charge and steric hindrance - DNA cannot bind Brf2 - 50 fold decrease in binding affinity Conclude that when Cys oxidised, complex assembly inhibited between DNA and brf2
28
What method was used to investigate RNA Pol 3 transcription in oxidative conditions? Conclusions?
RNA quantification + quantitative RT-PCR 1. RNA extraction from cells 2. Reverse transcription (RNA -> cDNA) 3. Quantitative PCR (specific oligo target) Brf2 = redox sensor, stops transcription in ox conditions Brf1 = independent of ox conditions (pol 1 transcription process intact)
29
What components make up TFIIIB?
TBP, Brf2, Bdp1
30
What is the structure of Bdp1?
- Long protein - Mainly disordered except for SANT domain - SANT shown to interact within TFIII complexes - N and C terminal domain shown to be important for activity
31
How does Bdp1 contribute to TFIIIB?
Bdp1 has disordered linker region that runs in minor groove of DNA Potentially contributes to TFIIIB-DNA stability Helps maintain complex at promoter for extended periods
32
What method was used to investigate TFIIIB stability?
Single molecule FRET - 2 fluorochrome each side of TATA box - When TBP binds at TATA box, bends DNA by 90 degrees - Brings fluorophores into close proximity, giving higher FRET signal
33
What were the conclusions following investigations of TFIIIB stability?
TBP alone at promoter has short half life (4.3 secs) Adding Brf2 and Bdp, higher FRET signals maintained after flushing suggesting much more stable complex (8+ min)
34
How does Pol III promoter opening in absence of ATP hydrolysis occur?
1. Bdp1 binds Cys residue, pushing subunit C34 2. Conformational change of structure allows DNA to open and move into active site
35
Why is cryo-EM useful in studying Pol 3 promoter opening?
Helps visualise mechanism of DNA opening by trapping different intermediate states
36
In the closed complex of Pol 3, where is DNA positioned?
DNA sits above the clamp and cannot enter the active site
37
In the open state of Pol 3, where is DNA positioned?
Open complex is defined by separation of the 2 DNA strands DNA positioned in active site
38
How does Pol III terminate transcription at poly(T) sequences in yeast?
1. Termination signal of 7 consecutive deoxythymidines (dT) on non template strand outside active site 2. C128 subunit of Pol 3 contains +ve residues that specifically recognise polyT 3. RNAP carries on until too many interactions between dTs and residues 4. Slows down and terminates transcription
39
Describe the conservation of residues involved in poly(T) termination?
Residues conserved in Pol 3 of over 15 species but not found in Pol 1 or 2
40
What experiment shows Pol III uses facilitated reinitiation?
Adding Pol III, DNA, TFs, and NTPs (not GTP) - Pol III engaged, starts ranscribing, stops when GTP needed Add heparin with GTP - Pol III can transcribe FL RNA - Heparin mimics DNA backbone - Blocks new assembly so any continued transcription must be from facilitated reinitiation from retained position - Transcription continues over time = confirmation
41
What is Maf1 activated in response to in yeast?
1. DNA damage 2. Oxidative stress 3. Growth -> stationary phase 4. Antibiotic treatment
42
How does Maf1 regulate Pol III under stress?
1. Maf1 becomes dephosphorylated 2. Translocates to nucleus to regulate Pol3 transcription 3. Maf1 binding prevents recruitment of TFIIIB, cannot initiate transcription

BIOC0020 (17 decks)

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