L1 : Pol III and Gene Regulation Flashcards
What is gene expression?
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
Different ways to control gene expression?
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
Draw a diagram showing different points of gene expression regulation
Describe features of nucleus
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
Compare euchromatin and heterochromatin?
Heterochromatin = densely packed and highly condensed
- Transcriptionally inactive (silenced)
Euchromatin = loosely packed and less condensed
- Transcriptionally active (expressed)
How do chromatin modifications allow events on DNA?
Condensed chromatin is repressive
How to relax/open it?
- Histone variants
- Chromatin remodellers
- PTMs on histone tails
What comprises each RNA polymerase and what do they transcribe?
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
Why do RNAPs have different numbers of subunits?
Some subunits and GTFs are specific for each system (eg. Rpb1 (1), A190 (2), C160 (3)) and some are shared
Some GTFs (Pol 2) are integral part of other Pol (1 + 3)
What is required for transcription?
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)
At mRNA genes (Pol 2)
- Some genes require activators (TFs)
- Co-activators (chromatin modifying enzymes and remodellers)
What is the pre-initiation complex?
Large multi-protein assembly that forms at the promoter region of a gene during the early stages of transcription
Positions RNA pol correctly and facilitates unwinding of DNA
What is the anatomy of an mRNA gene?
Core promoter, TSS, 5’ UTR, start codon, ORF, stop codon, 3’ UTR, termination
Core promoter contains sequence elements that GTFs can recognise in order to start transcription
What are key elements of the promoter?
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
What are the requirements to begin mRNA transcription (pol 2) successfully?
Core promoter DNA
RNA Pol 2 recruitment and positioning
Strand separation/melting
TSS selection
Note: highly purified Pol 2 inactive on double strand
How do GTFs (basal) provide these activities?
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
What is the process of formation of PIC?
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
When TBP bound, TFIIA stabilises interaction between TBP and DNA
Recruitment of TFIIB that assists in stabilisation, also recognising BreU and BreD elements
(Assists with promoter opening and TSS selection)
TFIIF assists in binding of RNAP2 to promoter bound TBP-TFIIB complex
(Also provides platform for later GTFs to bind)
TFIIE assists in promoter opening and recruitment of TFIIH
TFIIH contains ATP driven helicase enzyme
(Translocates along DNA and works to open promoter to present template strand for base pairing with NTPs)
What does pol 3 transcribe?
Short (>350 nt) and untranslated RNAs (tRNAs, 5S rRNA, U6 snRNA)
House keeping genes
What are the unique features of Pol 3 transcription?
- Extreme stability of TF3B
- No ATP hydrolysis required
- Termination at polyT
- Facilitated reinitiation
- Direct redox regulation by Brf2
What are the 3 promoter types of Pol 3?
Requires different PICs
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
What is TFIIIB composition depending on promoter type?
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
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
What is the role of the molecular pin in redox-sensitive DNA binding of Brf2?
- Molecular pin has conserved Cys that makes non-covalent interactions with DNA
- Oxidising cysteine increases size and gives charge
- No longer fits within cavity as electrostatically repulsed (-ve phosphate backbone)
- Assembly of complex is inhibited by presence of oxidised Cys
How was EMSA used to study redox-sensitive DNA-Brf2 binding?
Electrophoretic mobility shift assay
- Mutate Cys to aspartate
- Mimic sulfinic (charge and steric hindrance)
- Cannot bind Brf2
- Loses 50 fold affinity
Conclude that when Cys oxidised, complex assembly inhibited
What method was used to investigate RNA Pol 3 transcription in oxidative conditions? Conclusions?
RNA quantification + quantitative RT-PCR
- RNA extraction from cells
- Reverse transcription (RNA -> cDNA)
- Quantitative PCR (specific oligo target)
Brf2 = redox sensor, stops transcription in ox conditions
Brf1 = independent of ox conditions (pol 1 transcription process intact)
What components make up TFIIIB?
TBP, Brf2, Bdp1
