Transcriptional control of chromatin (L1-4) Flashcards
What is a promotor?
Cis acting DNA regulatory element that controls the initiation and control of transcription.
What is the strongest bacterial promotor ?
Consensus Sequence (-10 TATAAAT - -35 TTGACA)
What are CpG islands role in eukaryotic transcription?
Promotor elements eg TATA Box and initiator (Inr) are needed to initiate and control transcription in eukaryotes. However, many mammals lack these promotor regions or they are just not obvious, so CpG islands are needed. C residues in CpG islands escape methylation (become hypomethylated). Hypomethylation is associated with initiation of transcription.
What is methylation of CpG islands and what is it associated with?
When c residues followed by a G are methylated. Methylation causes transcription to be turned off. Therefore methylation is a negative thing for transcription in eukaryotes.
What are the regulatory regions on a eukaryotic gene?
Areas which turn transcription on or off.
>UAS and enhancers have binding sites for activator proteins, therefore activate transcription.
>URS and silencers have repressor binding sites.
UAS and URS are closer to transcription start site.
Reporter analysis is a powerful tool used for studying promotor action. What specific reporter gene was used to study what promotor in a mouse embryo?
Lac Z reporter used to study ure2 promoter. The more Lacz present will show how the promotor is working. Would be a linear relationship.
How many RNA Polymerases do prokaryotes and eukaryotes have?
P- 1
E- 3 (RNA POl 2 most important in transcription)
Describe the process of formation of pre initiation complex in eukaryotes.
- TFIID binds to TATA region of promotor. TFIID involves TBP and TAFs. Recruits TFIIB and TFIIA.
- TFIIA stabilises binding of TFIID. TFIIB recruits RNA POL 2 which is associated with TFIIF (helps RNA POL 2 bind).
- TFIIF simulates elongation and destablises non specific RNA POL 2 - DNA interactions.
- TFIIE recruits TFIIH.
- TFIIH is very important, see further card.
What subunits make up TFIID?
(first transcription factor with 13 subunits and has a trilobular structure)
TBP (TATA binding protein) and TAFs (TBP associated factors).
TBP is shaped like a molecular saddle.
TAFs promote interaction of TFIID with basal promotors and interact with activators.
The final transcription factor is TFIIH. What are the properties?
- TFIIH has ATPase activity which is required for promoter melting. TFIIH uses this energy to act as a helices to separate the strands. The ATPase is called XPB (or Ssl2)
- The CAK part of the factor contains the kinases which phosphorylate the C terminal domains of the RNA POL (CTD). CTD domain is phosphorylated whilst pol 2 starts transcribing.. This is promotor clearence.
what is the ATPase called in TFIIH?
XPB or Ssl2
Where are the C terminal domains located on the RNA POL?
The RPB1 subunit which is homologous to B’ on prokaryotic RNA Pol.
UAS/enhancers are used to increase transcription levels. What are the two classes of UAS/enhances
1.Common sequence elements- common means constant activation of promotors. These are located close to core promoter and bind activators that are abundant and constantly active.
2. response elements - bind factors whose activity is induced by stimuli.
What are 3 examples of ‘Common’ sequence elements and what factors bind to them?
1.GC Box - binds SP1 factor
2. Octamer - binds Oct-1 factor
3. CAAT Box - Binds NFY factor.
What are two examples of response elements, what do they bind and what is the factor induced by?
SRE- binds serum response factor which is induced by growth factors
HSE - binds heat shock factor which is induced by heat shock
what does it mean that eukaryotic activators are MODULAR?
The activation domain, DNA binding domain and flexible protein domains are independent units. They are separate. The activation
Activation domains are one of the domains in activators. Activation domains are characterised due to their amino acid composition. What are 3 examples of activation domains?
- Acidic patch - clusters of negative charged residues (Asp/glu) eg VP16
- Glutamine rich eg Sp1
- Proline rich eg Jun
What 2 main domains are activator proteins made up of?
Activation domain and DNA binding domain.
Two ways to analyse promotor regions?
Sequence comparisons and reporter analysis.
What do activators do- 3 different mechanisms?
- promote binding of additional activators
- increase rate of PIC formation
- stimulate release of stalled RNA pol 2.
How do activators increase rate of PIC formation?
- increase TFIID binding
- increase TFIIB binding
- increase RNA Pol 2 recruitment