17. Transcription factors

which GTF binds the TATA box

TBP (TATA bidning protein) within TFIID

6 RNAP II general transcription factors

TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH

what are the two main roles of TFIID

foundation for transcription complex assembly and nucleosome destabilization

which GTF has helicase and kinase activity

TFIIH

what happens to RNAP II’s CTD during promoter clearance

it is phosphorylated by TFIIH’s kinase activity

which factor recruits RNAP II to the promoter

TFIIB and TFIIF

what residues coordinate Zn in a C2H2 zinc finger

2 cysteines + 2 histidines

What DNA-binding motif is found in nuclear receptors

C4 zinc fingers

what is teh structure of a homeodomain

3 alpha-helices, 2 forma. helix-turn-helix, the 3rd inserts inot the major groove

what are the four possible domains in a TF

DN-binding, activation, dimerization, ligand binding

what does combinatorial control mean

different combinations of TF subunits regulate different genes

what is the role of coactivators

link activators to RNAP II complex and modify chromatin

what is the function of the mediator complex

bridge between activators and RNAP II: regulates initation rate

how far can enhancers act from the promoter

up to tens of kilobases; can be on different chromosomes

what does the yeast two-hybrid assay detect

interactions between a TF and DNA or between two proteins

How can promoter mapping be done

by creating a 5’ deletion series and measuring reporter gene expression

transcription initation complex

RNAP II + GTF bound: general transcription factors bound to promoter region

TFIID has two basic roles

1. foundation for the transcriptional complex

2. prevention of nucleosome stabilization

TBP is a component of the positioning factor

no matter which RNAP is transcribing → allows each type of polymerase to bind to its promoter

TAFs- TBP associated factors

determine whether or not TFIID stays at the promoter

TFIID complex

TATA binding proteins and 14 TBP associated factors (TAFs)

once TBP/TFIID bound

TFIIB can bin rate limiting step in transcription of many genes, proximal enhancers to TFIIB

• monomeric protein

• developmental regulation

TFIIB orients RNAP on DNA

TFIIB binds to bent DNA opposite and adjacent to TBP

• RNAP II will come in contact with TFIIB near RNA exit site and active site

TFIIE is DNA-dependent ATPase

probably necessary for generating the energy involved in melting a promter

TFIIH contains nine subunits

it has helicase activity and protein kinase activity

for transcription to occur, RNAPII must

be released from the promoter

TFIIH helicase activity will unwind DNA (with energy from TFIIE) and kinase activity is responsible for

phosphorylation of CTD tail → ctd tail detaches from RNAPII from TFIID

transcription pause released by

additional phosphorylation of CTD and other factors by CDK9

promoter clearance

mediator and GTFs are released

eu transcript overview

1. TFIID + TBP binds at TATA box → TFIID complex (14 TBP associated factors)

2. TFIIB binds → orients RNAP on DNA

3. TFIIF binds to mediator RNAPII → gather general factors

4. two large subunits of RNAPII interact with TFIIB

5. CTD tail (unphosphor) of RNAPII direct contact with TFIID

6. TFIIE and TFIIH binds

7. RNAPII is at the promter

8. TFIIH helicase activity unwinds DNA phosphorylates CTD tail

9. CTD tail detaches RNAPII from TFII

in majority of TATA-less promoters

first TBP has to be placed at the right spot in respect to the existing core element and transcription start site

initator element

TBP alone cannot bind to Inr-only promoters - requires the entire TFIID complex via TAFII-250 and TAFII-150

alternatively: TAFII-250 and TAFII-150 tether TBP to the initiator, allowing the rest of TFIID to assemble

TBP alone cannot bind to GC-only promoters

requires whole TFIID via TAFII-110 and Sp1 transcription factor or,

Sp1 binds GC boxes and interacts with TAFII-110, TAFII-250 and TAFII-150, anchoring TBP so the rest of TFIID can bind

purpose fo the yeast two-hybrid TF activity assay

to measure the activity of a transcription factor by detecting reporter gene expression controlled by its binding site

why is the host cell used in the assay lacking functional TF c

to ensure that any reporter gene activation comes from the introduced TF X

how can the yeast two-hybrid system help identify important domains of a transcription factor

by introducing mutations in the TF gene or its binding site and observing effects on reporter gene expression

proteins that directly influence RNAP binding

1. GTFs

2. TF = activators and repressors bind to DNA at proximal elements, enhancers/silencers

other proteins/complexes that affect transcription

1. co-activators and mediators

2. chromatin remodelling enzymes and other regulators

in order for protein to be transcription activator OR repressor, it;

1. has to be able to bind to DNA

2. activate or repress transcription

transcription factors are modular proteins

different domains - different funcitons

DNA binding domain

Dimerization domain

• one of the Zn finger

  • alpha helix in Zn finger C6

• leucine zipper part in bZip

• second helix in bHLH

transcription activation domain

• acidic

• glutamin rich

• proline rich

ligand binding domain

• in zn finger - C4 factors (steroid hormone is ligand)

DBD usually

interaction between a helices from the DNA binding
domain & major DNA groove  H-bonds, hydrophobicity and
Van der Waals (LDS) interaction

zinc fingers

protein structural motifs involving zinc ion coordination, often used for DNA binding, but also found in non-DNA-binding proteins

Three types of zinc fingers

1. C2H2 zinc finger (classical form found in >1000 transcription factors)

2. C4 zinc finger (nuclear receptors, often for dimerization)

3. C6 zinc finger (found in yeast transcription factors like GAL4)

homeodomain proteins

drosophila - group of homeotic genes, when altered lead to transformations of one body part for another (legs for antenna) = homeotic transformations (transitions)

the locations and developmental roles of homeotic genes are

strikingly similar across many species

the gene order in clusters corresponds to the order of

expression of genes and their function along the anterior-posterior body axis

homeotic genes mostly code for

transcription factors - involved in controlling development - establishment of spatial pattern

leucine-zipper proteins bZIP

• hydrophobic aa leu in every 7th position fo the c-terminal region for dimerization

• forms alpha-helix with leucine residues

• second alpha helical regino is invovled in DNA binding

• form homodimers and heterodimers through a coiled structure maintained by hydrophobic forces

helix-loop-helix

• hydrophobic residues on one side

• non-helical loop of polypetide chain separates teh two alpha helices from each monomeric protein, second alpha helix responsible for DNA binding (basic charge)

• form heterodimers