Transcription Machinery Flashcards
(20 cards)
List the eukaroytic RNA polymerases and their function
RNA Polymerase
- RNAP I: synthesises rRNA precursor (28S, 18S and 5.8S), responsible for 50% of RNAP activity.
- RNAP II: synthesises mRNA
3.RNAP III: synthesises small non-coding RNAs that encode RNAP, tRNAs and 5S rRNA
List the subunits of RNAP and names of eukaryotic subunits
RNA Polymerase
Beta, Beta prime, Alpha (x2), Omega
RPB1 (Beta prime), RPB2 (Beta), 3 and 11 (Alpha), RPB6 (Omega)
Explain what the CTD is.
Carboxy Terminal Domain
Unstructured domain of RNAP II with tandem repeats of seven amino acids.
Tends to be close to the core promoter, CTD length seems to inversely related to gene density.
Evidence of CTD-CTD interactions.
Post-translational modifications are necessary for transcription.
CTD acts as landing site for transcription factors involved in initiation, elongation, capping, splicing, cleavage and polyadenylation.
Name factors involved in the PIC.
Pre-Initiation Complex
General transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH), Mediator and co-activators
List properties and functions of TFIID.
TFIID
Subunits include TATA binding protein (TBP), 13-14 TBP associated factors (TAF).
TBP and some TAFs make sequence specific contacts with promoter DNA.
TAFs recognise modified histones.
TBP and TAFs are targets for activator proteins.
TFIID is a large multi-protein complex, almost the same size as RNAP II.
List properties and functions of TFIIA.
TFIIA
Contains 3 subunits, but not required for basal transcription.
Co-activator, needed for transcription activation in vivo.
Stabilises TBP-TATA complex by inhibiting TBP dimer formation and non-productive TAF1 and TBP interactions.
List properties and functions of TFIIB.
TFIIB
Single polypeptide.
Makes contact with B recognition Element (BRE), which is a sequence within the core promoter.
Stabilises TFIID-Promoter complex.
Recruits TFIIF holoenzyme.
Interacts with activator proteins
TFIIB and RNAP II are responsible for start site selection.
List functions and properties of TFIIF.
TFIIF
Heterotetramer.
Enters the complex as a holoenzyme with RNAP II.
Stimulates elongation rates by supressing pausing.
Stabilies RNAP II binding to TFIID-TFIIB-complex.
Recruits TFIIE and TFIIH.
Involved in start site selection and promoter escape.
List functions and properties of TFIIE.
TFIIE
Heterotetramer.
Binds to RNAP II in solution and may therefore enter complex with RNAP II-TFIIF.
Interacts directly eith TFIIB, TFIIF, RNAP II and promoter DNA.
Stimulates enzymatic activity of TFIIH ( ATPase, CTD kinase and helicase).
Forms a bridge between the other GTFs.
List the properties and functions of TFIIH.
TFIIH
Largest and most complex GTF.
Contains 10 protein subunits, with some enzymatic activity.
Cyclin-dependent kinase (CDK7), phosphorylates Ser5 of the CTD.
ATPase involved in transcription initation and promoter escape.
Helicase required for promoter melting.
Involved in transcription-coupled nucleotide excision repair.
List properties and functions of Mediator.
Mediator
Required for PIC formation.
Conductor in transcription.
Activator recruits mediator, mediator recruits everything else.
Forms a bridge between activators and transcription machinery.
Forms a scaffold of PIC assembly.
Describe the core promoter.
Core Promoter
Region around transcription start, between -30bp to +30bp.
Associated with regulatory proteins and either enhancers or PPEs.
Can be focused, dispered or mixed.
Can be TATA-containing or TATA-less.
Sequences that may be found in focused core promoters.
Focused core promoters
BRE (upstream or downstream)
MTE
DPE
TATA
DCE (1, 2, 3)
Describe focused promoters.
Focused promoters
Regulated.
Nucleosomes not precisely positioned.
Insulator overlaps transcription start.
Often TATA-containing.
Methylation and acetylation goes across the whole promoter.
Have epigenetic marks when active (H3K4me3 and H3K27ac).
Describe CpG Islands.
CpG Islands
Associated with dispersed promoters.
0.2-2kb regions that are rich in unmethylated CpG dinuleotides.
50% of CpG islands associated with core promoters.
Contain multiple weak insulator elements.
Lack TATA and DPE elements.
Enriched for TF binding sites, such as Sp1, Nrf-1, E2F and ETS.
House keeping genes contain single CpG islands.
Advantages of ATSS.
Alternative Transcription Start Site
Allows for complexity in gene expression.
50-100 bp difference.
Variation in protein according to need.
Alternative 5’-UTR or exons.
Describe the two tracks for PIC assembly.
Pre-Initiation Complex
- TBP binds tightly to the TATA box and lobe C of TFIID binds tightly to downstream elements (Inr, DPE, etc).
- Either binding tightly to the TATA box OR downstream elements. This causes TBP or lobe C to be moved upstream or downstream (respectively).
Describe the ‘drive, neutral, park’ formations.
Drive, Neutral and Park
Drive - active transcription. Occurs when TFIIH binds.
Neutral - paused. Occurs when TFIIE binds.
Park - transcription inhibition. TFIID makes contact with promoter elements which constrains TBP and lobe C, prohibiting transcription when RNAP II binds.
What is the advantage of TATA-DBE promoters?
TATA-DBE promoter (track 1)
Regulated (need activator proteins)
Binding may be quicker
More controlled
Describe the scaffold complex and its advantages.
Scaffold Complex in GTF Recycling
Quicker reinitiation
Allows for RNAP II to go off and transcribe other genes
