Lecture 7: Eukaryotic Gene Expression Transcription Flashcards
What are the differences between eukaryotic and prokaryotic gene expression?
Prokaryotic vs Eukaryotic
polycistronic monocistronic
cytoplasmic nuclear
coupled transc-transl not coupled
single RNA polym. 3 diff RNA polym
uninterrupted interrupted (splicing)
What does polycistronic mean?
more than 1 proteins coded for in one mRNA transcript
what do the three RNA pol. transcribe?
- rRNA
- mRNA (via hnRNA)
- tRNA
What are the three factors that can interact with RNA pol2 to modify transcription?
- GENERAL FACTORS: form basal transcription apparatus with RNA pol2 itself
- UPSTREAM FACTORS: DNA-binding, recognising short sequences, unregulated
- INDUCIBLE FACTORS: recognised to control gene expression in a stimulus specific manor
What is the core promoter?
‘minimal DNA elements required for accurate transcription initiation by RNA polymerase 2’
What are the accessory factors of RNAP2? How many are there and what are they?
6 general initiation factors; TF2D, TF2A, TF2B, TF2F, TF2E, TF2H
RNAP2 + these factors = basal transcription apparatus
What is the pre-initiation complex (PIC)?
“The preinitiation complex is a complex of approximately 100 proteins that is necessary for the transcription of protein-coding genes in eukaryotes and archaea”
What is TF2D composed of? What is its purpose?
Assembly of a pre-initiation complex (PIC)
Promoter recognition:
- TF2D = TBP + TAFs
- TBP (TATA-binding protein)
- TAFs (TBP-associated factors)
This is used to position RNAP2 in the right place. Also binds DNA in minor groove (binds DNA ~80 degrees)
What is the role of TF2D (TBP + TAFs) in PIC assembly?
- recognise core promoter (TATA box)
2. Recognise core promoter (no TATA box)
What is the role of TF2A in PIC assembly?
Stabilise TBP and TAF binding
What is the role of TF2B in PIC assembly?
Aids RNAP2/TF2F recruitment and transcription start site selection
What is the role of TF2F in PIC assembly?
- Brings RNAP2 to promoter
2. Breaks non-specific RNAP2-DNA interactions
What is the role of TF2E in PIC assembly?
- Recruits TF2H
- Regulates TF2H helicase
- ATPase and kinase activities
What is the role of TF2H in PIC assembly?
- Aids promoter melting (dsDNA to ssDNA)
2. phosphorylation of CTD for promoter clearance
How many repeats are there in RNAP2 C-terminal domain (CTD)? What amino acids are subject to phosphorylation?
52 repeats of YSPTSPS in mammals
Ser2 and Ser5 subject to phosphorylation
What is unphosphorylated RNAP2A used for
Initiation. Interacts with general initiation factors.
What is phosphorylated RNAP2O used for?
Elongation.
- TF2H (and others) is CTD kinase
- allows dissociation of first TF2B and E then TF2H
- TF2F remains associated
What are the roles of RNAP2 CTD?
- Splicing
- Capping
- Addition of polyA tail
How is transcription activated and repressed?
- Cis-acting control elements (DNA)
a. promoters
b. enhancers/repressors - Trans-acting transcription factors
(protein)
a. constitutively expressed or induced
b. two domain (DNA binding and
activation domains)
Where is TATA box located? Where is the CAAT box located? Where is the GC box located?
What happens as a result of mutations in these regions?
TATA box = -30
CAAT box = -75
GC box = -90
Mutation results in far lower transcription levels
How do promoters ‘mix and match’
They contain different numbers of TATA, CAAT, GC boxes, and other elements
“TRANSCRIPTION OF GENE A CONTROLLED BY ACTIVATORS 1, 2, 3, and 4. TRANSCRIPTION OF GENE B CONTROLLED BY ACTIVATORS 2, 4, and 6”
How can a combination of control elements activate transcription?
Only when activator proteins are present (and potentially activated)
What are the two domains of TFs?
- DNA-binding domain
- recognise target sequence in enhancers
or promoters
- recognise target sequence in enhancers
- Activation domain
- function to influence transcription by
contacting basal apparatus
- function to influence transcription by
What are the protein structure motifs in DNA binding?
1. Helix-turn-helix (e.g., homeodomain
containing proteins)
2. Zinc finger (e.g., steroid receptors)
3. Helix-loop-helix (e.g., c-Myc)
4. Basic region-leucine zipper
(e.g., Fos/Jun = AP1What is a transcriptional Co-activator?
“Does not bind to DNA itself, but bridges between the activator and the basal transcriptional apparatus”
What does CRE, CREB, and CBP mean?
- CRE = cyclic AMP response element
- CREB = CRE binding protein
- CBP = CREB binding protein
What is an enhancer?
They are “distance, orientation and position independent” from the promoter
What is transient transfection?
1. Obtain genomic clone of gene - obtain
'promoter region'
2. Obtain reporter vector e.g.,
a. CAT
b. Luciferase
c. beta-gal
d. GFP
- sub clone promoter fragment
3. Transfect into cells
a. CaPO4
b. DEAE-dextran
c. Lipid
d. Electroporation
- measure reporter under test conditions
