Fundamental Part of Eukaryotic Transcription
what does activators do
what needs to be recruited
what is the main rate limiting step
- opens up genomic DNA
- RNA polymerase 2 and general transcription factors
- Pre-Initiation Complex
Chromatography
- seperates mixtures based on x such as y
- three steps/parts
- X: Technique to separate a mixture of molecules based on differences in their chemical properties. Y: for example: differences in size, charge, solubility
- a) Usually involves taking your sample (mixture of molecules) in liquid phase
b) And passing it through some type of
resin/beads by gravity, centrifugation, or
adding more liquid
c) that will allow separation of the more complex mixture into smaller mixtures of molecules in discrete pools or fractions
Polymer beads with - charged groups
Ion Exchange chromatography
4 steps
- Pass sample through
resin/beads that are charged - In cation exchange chromatography, beads are negatively charged
- negatively charged samples are
repelled by beads and enter the
earlier collected fractions - positively charged samples are
attracted to the beads and either
remain stuck to beads or enter
the later collected fractions
4 steps of Isolation of the 3 Eukaryotic RNA polymerases
1) Starting material: Prepare soluble protein extracts from the nuclei of
sea urchin embryos (also used rat liver) – these organisms/tissues
were chosen because it was easy to get large amounts of nuclear
material from them
2) Use anion exchange chromatography column (DEAE-sephadex
resin), load extract on this column
3) Use a gradient of salt (increasing concentration of Ammonium
sulphate) to separate fractions containing more purified
complexes/mixtures.
4) Final assay: test whether each fraction has the ability to incorporate
radioactively labeled nucleotides into actively synthesized RNA
(Uridine triphosphate - UTP).
* levels of radioactivity can be detected and measured
- how were the three eukaryotic RNA polymerases discovered
- This discovery was achieved by chromatography of what and identifying what
- This discovery set the stage for understanding what
- The three eukaryotic RNA polymerases were discovered by
purification of enzymatically active fractions - This discovery was achieved by chromatography of soluble nuclear proteins and identifying which fractions were capable of incorporating radioactive nucleotides
- This discovery set the stage for understanding how
transcription initiation and elongation works
- what control the rate of transcription of protein-coding genes
- General transcription factors (GTFs) are necessary
- What are the GTFs
- Mechanisms controlling the assembly of the RNAPII transcription PIC essentially control the rate of transcription of protein-coding genes
- General transcription factors (GTFs) are necessary for accurate transcription initiation in vitro
-> TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, other components of RNAPII
-> Multi-subunit Mediator complex (enhances transcription but not required in vitro)
- how do GTFs assemble
- These factors also help
- Eukaryotic general transcription factors are highly conserved (most eukaryotes have them)
- GTFs assemble on the core promoter in an ordered fashion
to form a PIC, which accurately recruits RNAPII to the transcription start
site (TSS) - These factors also help to melt the DNA duplex (unwind 2 strand DNA) , so that the template strand can enter the active site of RNAPII
Development of a minimal system for accurate transcription in a test tube: the in vitro transcription assay experiment
5 steps
- Use crude cell-free extract (this extract was called S100)
- S100 is actually a cytoplasmic extract with some leakage of nuclear proteins during the preparation, but very little RNA polymerase II
- Add purified RNA polymerase II
- This is needed to support transcription, but purified RNA polymerase II on its own will not
achieve accurate transcription - Add DNA template (originally adenovirus major late promoter DNA)
- this was a well studied virus at the time, and the region near the transcription start site was accurately known
- Follow incorporation of radioactive nucleotides over time
- Radioactivity enables the labeling of the nascent mRNA transcript
- Gel electrophoresis, autoradiography
- Gel electrophoresis separates the RNA by size, and autoradiography captures the energy emission from radioactivity, which can be transferred to film or a storage screen (essentially takes a photograph)
assembly of the GTFs and RNA polymerase II occurs in x and what does this lead to
- Using these type of experiments (and others) it was determined that the
assembly of the GTFs and RNA polymerase II occurs in a specific sequence - This sequence leads to the formation of the pre-initiation complex
- Promoter elements
recognized by TFIID - TBP (binds to, interacts with, bends)
- Subunits of TFIID
- TATA box (TBP)
* (TAF1/2)
* (TAF6/9) - binds to promoter element
* interacts with minor groove of
DNA duplex
* bends the helix facilitating
TFIIB attachment
- binds to promoter element
- TBP – TATA box binding protein
* TAF1-13
- TBP – TATA box binding protein
- TFIIF/RNAPII
- TFIIA
- TFIIA
- TFIIH
- is positioned over top of the TSS
- required for stability of TFIID
and TFIIB components in vivo - complex binds to create
docking site for TFIIH - TFIIH complex is a helicase and
uses ATP hydrolysis to unwind
duplex, exposing the template
strand. TFIIH also has important kinase
activity that phosphorylates the Cterminal Domain (CTD) of the large
subunit of RNA polymerase II
Dual roles of TFIIH at initiation of transcription
- ATPase activity helps with melting/opening the DNA to allow for
transcription to begin - Phosphorylation of C-Terminal Domain (CTD) of the large subunit of
RNA Polymerase II. *The CTD is composed of tandem repeats of a conserved heptad amino acid sequence
YSPTSPS.
