Lecture 17 (RR4): Proteins required to initiate eukaryotic transcription Flashcards
TATA box
- Sits very proximally at around -35
- TATA sequence directs transcription at the promoters of some protein coding genes
Technique used to understand the proteins that were necessary for transcription (Run off assay)
To understand the collection of proteins that were necessary for transcription in vitro:
- They used a promoter containing DNA template (like a plasmid) that is known to be a target for Pol II transcription and has defined RNA that will be synthesized.
1) Often use linearized DNA molecules. Place into a test tube with the appropriate conditions and you allow the proteins to sit down on the promoter and carry out their normal functions.
2) They would start transcription at the predicted start site and then synthesize an RNA product until it came to the end of it.
3) After this, the RNA polymerase would fall off the template and could reinitiate back at the beginning of the promoter.
- If you did the whole reaction with a series of labelled rNTPs, the RNA transcribed that is synthesized in that reaction would be labelled and you could detect it using any method (often radioactivity).
- They were able to separate out whole cell or nuclear extracts and identify the key proteins that when you mix them together in the presence of a competent DNA template (that has a RNA polymerase recognizable template) it would carry out a transcription reaction.
The whole point of the run off assay is:
- To determine what proteins (TFs) are needed for transcription to work.
1. Place a linearalized DNA, rNTPs…
2. When u have the right combination of proteins you will be able to synthesize RNA.
3. Reconstruct and see if you restored transcription reaction.
Separation of general transcription factors using liquid chromatography
Example of how this kind of work was carried out initially = Done using chromatography.
* Nuclear extracts were run across a liquid chromatographic column and separated out into different fractions.
* When these fractions were separated, it was only when you combined specific fractions that you could restore transcription in vitro based on RNA polymerase II.
* The major factors identified during this process included: TFIIA (transcription factor, class two), TFIIB, TFIID, TFIIE, TFIIF, TF11H. When all of these were combined, they would work together with polymerase II to form a RNA product. You could detect this RNA with your method of choice (usually run on assess).
TFIID
- Identified through many different manners
- One of the most important
- Large protein that interacted specifically with the TATA box region by virtue of one of its subunit TBP.
- TBP is one subunit of a large multiprotein complex.
- These other subunits that make up TFIID are referred to as TBP associated factors.
TATA-box Binding Protein (TBP)
- Small protein
- The conserved C-terminal domain of TBP binds to the minor groove and distorts the double helix (binding the minor groove creates a kink).
- Looks like a saddle and has extended stirrups that interact with the DNA.
- Many promoters are “TATA-less”, but still require TBP for initiation.
- TBP is also required for efficient Pol I and Pol III transcription.
- TBP and TFIID are really important for binding the TATA box but they are also required in in vitro transcription reactions of sequences that dont even contain the TATA box sequence. Therefore, they are required for all transcription/in vitro transcription despite the fact that it doesn’t necessarily even have to bind the TATA box element.
Why are TBP and TFIID necessary for transcription on TATA-less promoters?
- TFIID and TBP were required for transcription on TATA-less promoters because some subunits interacted with other proximal elements around the transcriptional start site.
- Some examples: TAF2 binds to the initiator, TAF6 interacts with downstream promoter elements.
RNA Polymerase II transcription
1) One of the early steps in the initiation of transcription includes TFIID binding to a promoter that contains a TATA box (or not).
2) By doing this, it will interact tightly with the DNA and add in other transcription factors (TFIIA and TFIIB) - could be by virtue of the kink.
3) When TFIIA and TFIIB join this complex with TFIID it becomes stabilized. These (A,B and D) can be referred to as an upstream complex.
4) RNA pol II can interact with this critical complex, it has an inherent capacity of connecting with TFIIB → TFIIB is what brings it into that complex with the help of TFIIF. These two like to be together (TFIIF and RNA polymerase).
5) This gives rise to the core pre initiation complex (PIC). RNA polymerase is now appropriately recruited to the promoter that it will activate downstream.
6) Another two transcription factors come in, TFIIH and TFIIE. Once these come into the complex, the complex becomes closed and is stabilized on that DNA.
7) In the presence of ATP or dAPT, the DNA will be melted and open up. Allowing RNA pol II to interact with the template strand and be ready to switch into an elongation phase.
8) Elongation phase will occur when RNA polymerase II is given rNTPs and immediately it switches, it goes from a pre-initiation to an elongation phase.
- TFIIH is responsible for melting/unwinding the DNA. It uses ATP.
- The subunits of TFIIH that are responsible for this are two DNA helicases which are critical for transcription.
TFIIH
The final fraction of TFIIH contained at the time 9 different polypeptides.
* The two large DNA helicases are XPB and XPD.
* The surprise about TFIIH was not just about its ability to open the complex but when we identified that XPD and DPB are DNA helicases that are absolutely necessary for nucleotide excision repair.
* P44 is also involved in DNA repair.
* A last protein that was only identified later was TTDA-p8 also critical for DNA repair.
* Cdk 7, cyclinH and MAT1 are involved in cell cycle control.
* Cdk 7 is crucial in order to activate all the main cdks in the cell that drive the cell cycle forward. Also, responsible for the 1st phosphorylations that are put onto the carboxy terminal domain of the large subunit of DNA polymerase II. Plays an essential role in every transcription reaction that is based on RNA POL II that take place in our cells.
Two types of transcription factors
- Basal transcription factors can bind to the core promoter region of genes and help RNA polymerase bind.
- Specific transcription factors bind to regions outside the promoter to enhance or repress expression.
Xeroderma Pigmentosum
- TFIIH is the only general/basal transcription factor that has ATP-dependent enzymatic activities.
- TFIIH contains two DNA helicases involved in Xeroderma pigmentosum (human disease → XP). You can rescue the deficiencies by putting in wild-type TFIIH.
- Xeroderma pigmentosum patients have a faulty nucleotide excision repair (NER) system
- Some patients have defects that are much more severe.
These patients are predisposed to cancer because when they go outside and are exposed to UV light their cells cannot repair the thymine dimers that arise.
DNA Repair
- The repair of various regions is not the same in all of the genome.
- Heavily transcribed regions are repaired more effectively.
- Lesions are repaired rapidly around the transcriptional start sites.
- TFIIH contributes/ is required to transcription-coupled DNA repair.
Formation of a pre-initiation complex
1) Recognition of the DNA region around which RNA Pol II will be recruited to activate the downstream gene. This is dependent on the association of TFIID with those regions.
2) TFIID will then recruit in a number of other general transcription factors (required for all class II transcription).
3) TPB is the core DNA binding subunit of TFIID (maintains many other subunits that help with TPB).
4) By recruiting all the other general transcription factors, you end up setting up a preinitiation complex that is very stable on a given promoter.
5) Only when provided with an ATP dependent source, you will actually be able to harness those helicase activities of TFIIH and melt the DNA to create an open complex.
6) This is also going to be associated with a presence of rNTPs, which will allow RNA Pol II to leave the promoter and switch to a phase that is more typical of elongation.
7) RNA polymerase becomes heavily phosphorylated and this phosphorylation is dependent on TFIIH cdt activity that resides in the subunit cdk7, cyclin H and MAT1. This phosphorylation of the carboxy domain seems to be an important switch that allows RNA polymerase to leave the general transcription factors at the promoter and start to move away (promoter clearance).
8) The transcription factors will then dissociate and RNA pol II will transcribe up to 100 nucleotides until it stops again (usually near the first nucleosome).
** REMEMBER: RNA pol II is phosphorylated on a carboxy terminal domain (CTD) in all the situations where transcription is highly active (we saw it in the chromosome puffs).
What is the kinky tail and why is it important?
- The kinky tail is a series of repeats 52 (in humans) if you eliminate this you die.
- It is important because you need the CTD and residues in the CTD because it signals molecularly the switch from transcription initiation to the next step.
