proteins required to initiate eukaryotic transcription

general transcription factors

minimum number of factors required to combine with polymerase II and cause transcription

adenovirus major late promoter (ADMLP)

• well-studied

• strong promoter (with TATA box)

• can be used as a promoter to separate general transcription factors using liquid chromatography

separation of general transcription factors using liquid chromatography

1. segment of DNA with promoter and digested end is transcribed in vitro with proteins (nuclear or whole cell extract) with labelled NTPs

2. RNA polymerase runs off

3. RNA products can be separated and quantified by acrylamide gel electrophoresis followed by autoradiography (or another means of detection)

general transcription factors identified from nuclear extract using ion exchange chromatography

• TFIID

• TFIIA

• TFIIB

• TFIIE

• TFIIF

• TFIIH

TATA-box binding protein (TBP)

• domain of TFIID

• conserved C-terminal domain binds to the minor groove and distorts the double helix

• required for initiation of many TATA-less promoters

• also required for efficient Pol I and Pol III transcription

need for TFIID even in TATA-less promoters

explained by interactions between TAF2 and TAF6 on TFIID and Inr and DPE on DNA (respectively)

assembly of pre-initiation complex

1. TBP region of TFIID binds to TATA box and causes kink in DNA

2. TFIIA and TFIIB associate with TFIID on DNA, forming upstream promoter complex

3. TFIIF and Pol II associate with upstream promoter complex, forming core pre-initiation complex (arrive together because of strong affinity)

4. TFIIE and TFIIH (with TFIIH kinase) arrive, forming closed PIC

5. in the presence of energy (ATP) and rNTPs, pre-initiation complex opens

TFIIH

• the only general/basal transcription factor that has ATP-dependent enzymatic activities

• contains two DNA helicases involved in Xeroderma pigmentosum (XPB and XPD)

Xeroderma pigmentosum

faulty nucleotide excision repair system

transcription-coupled DNA repair

heavily transcribed regions are repaired more effectively because DNA repair factors are associated with general transcription factors