table 1 complexes involved in rnapii pic assembly. protein complex functions rnapii 12 subunits;...

Table 1Complexes Involved in RNApII PIC assembly.Protein complex FunctionsRNApII 12 Subunits; catalyzes transcription of all mRNAs and a subset of noncoding RNAs including snoRNAs

and miRNAsTFIIA 2–3 subunits; functions to counteract repressive effects of negative cofactors like NC2; acts as a

coactivator by interacting with activators and components of the basal initiation machineryTFIIB Single subunit; stabilizes TFIID-promoter binding; aids in recruitment of TFIIF/Pol II to the promoter;

directs accurate start site selectionTFIID 14 subunits including TBP and TBP Associated Factors (TAFs); nucleates PIC assembly either through

TBP binding to TATA sequences or TAF binding to other promoter sequences; coactivator activity through direct interaction of TAFs and gene specific activatorsTFIIE 2 subunits; helps recruit TFIIH to promoters; stimulates helicase and kinase activities of TFIIH; binds

ssDNA and is essential for promoter meltingTFIIF 2–3 subunits; tightly associates with RNApII; enhances affinity of RNApII for TBP-TFIIB-promoter

complex; necessary for recruitment of TFIIE/TFIIH to the PIC; aids in start site selection and promoter escape; enhances elongation efficiency

TFIIH 10 subunits; ATPase/helicase necessary for promoter opening and promoter clearance; helicase activity for transcription coupled DNA repair; kinase activity required for phosphorylation of RNApII CTD; facilitates transition from initiation to elongation

Mediator At least 24 subunits; bridges interaction between activators and basal factors; stimulates both activatordependent and basal transcription; required for transcription from most RNApII dependent promoters

SAGA 20 subunits; interacts with activators, histone H3, and TBP; histone acetyltransferase activity; deubiquitinating activity

Trf1 TBP related factor identified in Drosophila; upregulated in CNS and gonads during development; can bind TATA sequences; mostly found at RNApIII dependent promoters as part of TFIIIB but also required at a

subset of RNApII dependent promotersTrf2 TBP related factor identified in all metazoans; cannot bind TATA sequences; important for histone gene

expression in DrosophilaTrf3 TBP related factor identified in vertebrates; can bind TATA sequences; important for differentiation of

muscle cells in mammals and for haematopoietic cell development in zebrafish.TFIIS 1 subunit; stimulates intrinsic transcript cleavage activity of RNApII allowing backtracking to resume RNA

synthesis after transcription arrest; stimulates PIC assembly at some promotersNC2 2 subunits; binds TBP/DNA complexes and blocks PIC assembly; can have both positive and negative

effects on transcriptionMot1/bTAF1 1 subunit; induces dissociation of TBP/DNA complexes in ATP dependent manner; can have both positive and negative effects on transcription

Questions:

1. How does the PIC decide where to form?2. Why isn’t the PIC sufficient for efficient gene expression?3. How are PIC assembly and transcriptional activation controlled?

PIC = Pre-initiation complex; this is the “business end”of the transcription apparatus.

Image from euchromatin.org

Gnatt et al, (2001) Science, vol 292, 1876-1882

Eukaryotic Basal DNA elements:

TATA element (TATA)Initiator element (INR)Downstream Promoter element (DPE)TFIIB recognition element (BRE)

Any given eukaryotic promoter will haveone or more of these elements, but seldom all of them. The PIC can thus berecruited to different promoters in differentWays.

TFIID is a major player in transcriptional initiation.

Thought to nucleate PIC assembly through TBP binding to DNA.

Its composition can change depending on which subunits are included (see next fig).

About 10% of genes are dependent on SAGA rather than TFIID. SAGA TFIID are probably homologous. SAGA also includes TBP.

Is TFIID required for every round of transcription?

TFIID

Cler et al, (2009) Cell Mol Lif Sci, vol 66, 2123-2134

TFIIH

TFIIH has three enzymatic activities:2 helicases (for promoter opening and for DNA repair)1 kinase (for phosphorylating CTD of RNA pol II)

Involved in transitioning from initiation to elongation.

Keep in mind:

Basal transcription factors (ie PIC) don’t transcribe chromatin templates.

Basal transcription factors don’t respond to activators.

This raises the level of complexity, and provides avenues for regulation,in two ways.

See upcoming lectures by:

Whitehouse (chromatin)DiGregorio and Pertsinidis (activators and regulatory modules)

Mediator

Basal Factor or not? Not required for basal activity in vitro, but it canstimulate this activity.

It has been proposed to stimulate phosphorylation of the CTD of RNA pol II (see Shuman lecture).

Its composition can change depending on which subunits are included (again……).

Might also act by stimulating reinitiation.

D’Alessio et al, (2009) Mol Cell, vol 36, 924-931

SAGA

Our first mention of histone modifying activity.

As mentioned earlier, can substitute (and might be related to) TFIID.

Like Mediator, it can interact with both activators and TBP, acting asa bridging molecule.

Baker and Grant, (2007) Oncogene, vol 26, 5329-5340

TFIIS

Originally characterized as an elongation factor, however more recentlyshown to play a role in initiation and PIC formation as well.

Prather et al, (2005) Mol Cell Biol, vol 25, 2650-2659

Repressors: Mot1/bTAF1 and NC2

These proteins affect TBP/DNA binding by either blocking PIC assemblyor inducing dissociation of TBP from the DNA.

Counter-intuitively, these can act as activators by preventing or reducingin appropriate binding of PIC to cryptic promoters, thus makingmore PIC available for activation of appropriate promoters.

van Werven et al, (2009) Genes and Development, vol 22, 2259-2369

D’Alessio et al, (2009) Mol Cell, vol 36, 924-931

Model of “Regulated Recruitment”

Potential mechanisms of transcriptional activation:

1. Regulated Recruitment: proteins bind to DNA and recruit an activepolymerase complex to the promoter.

2. Polymerase Activation: proteins bind to DNA and activate a pre-bound, inactive polymerase complex, thus initiating transcription.

3. Promoter Activation: proteins bind to DNA and induce a conformational change in the DNA, twisting it into a active state that allows a transcriptionally active polymerase can bind.

(Figures and concepts taken from Ptashne, M. and Gann, A. (2002) Genes and Signals. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA.)

table 1 complexes involved in rnapii pic assembly. protein complex functions rnapii 12 subunits;...

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