Transcription in eukaryotes •RNA Polymerases •Promoters •General Transcription Factors

Model of yeast RNA polII with RNA-DNA hybrid in the active site

Eukaryotes have 3 different RNA polymerases

Amanita phalloides

a-Amanitin

Sensitivity or RNA Pols to a-amanitin

Structure of RNA Polymerases •all 3 are large multimeric proteins (500-700 kDa) •all have two large subunits with sequence similarity to b and b’ of E. coli. Therefore, catalytic sites of three RNA Pol I, II and III may be conserved.

Epitope tagging to Identify RNA Pol II subunits

Subunit structure of yeast RNA polymerase II

RNA Polymerase II Structure Summary •Yeast Pol II enzyme consists of 12 different poly- peptides (RPB1 - 12). •Core subunits are RPB 1, 2 and 3. •RPB1 and 2 are orthologues of E. coli RNA Pol b’ and b. •RPB1 has DNA binding site and has carboxy-terminal domain (CTD). CTD has repeating heptad PTSPSYS - hydrophilic sequence. •5 of 7 aa’s of heptad have ‘-OH’ groups that can be phosphorylated

RNA Polymerase II Structure Summary Continued •RPB2 (b ortholgue) is at or near active site. •RPB3/11 may function as orthologue of a-dimer of prokaryotic RNA Pol •RPB5, 6, 8, 10 and 12 are shared between RNA Pol I, II, and III •RPB4 and 9 are non-essential RNA Pol II subunits

Structure of Prokaryotic and Eukaryotic RNA Pols

Eukaryotic Promoters •3 classes of eukaryotic promoters: I, II and III - transcribed RNA Pol I, II, and III respectively. •Class I promoters not well conserved in sequence amongst different species but general architecture is: consists of a core element and an upstream promoter element (UPE). •Some Class III promoters have promoter sequences wholly within the gene. Another type of Class III promoter resembles Class II promoters. •Class II promoters have up to five conserved elements.

First consensus sequence from lining up several eukaryotic promoters: TATA box

Generic class II promoter TFIIB recognition Downstream Promoter element

Transcription Factors •Eukaryotic RNA Pols cannot bind to promoters on their own (unlike prokaryotic RNA Pols). •Transcription factors are required for eukaryotic RNA Pol binding. •General transcription factors allow binding of RNA Pol binding to promoters and a basal level of transcription. •Gene-specific factors stimulate transcription further (or repress it) and allow fine regulatory control.

Six general transcription factors: TFIIA, B, D, F, E and H initiate transcription. •Gel shift assays showed that TFIID, A and B can form a complex independently on DNA (i.e. without RNA RNA Pol) •TFIIF binding to DAB complex is dependent on RNA Pol. Therefore, RNA Pol and TFIIF are needed together to join preinitiation complex. •Binding of TFIID to the TATA box occurs first; This event is required for binding of all of the other TFs. •In vitro, TFIIA is not critical for preinitiation complex formation.

Model for DABPolF complex formation

TFIID •TFIID consists of a TATA-box binding protein (TBP) and eight TBP-associated factors (TAFIIs). •TBP is well conserved between different organisms. •C-terminal 180 amino acids of TBP is sufficient for binding to the TATA box. •TBP interacts with the minor groove of the TATA box -unusual. •TBP also functions with Pol I and III. It is a universal transcription factor.

TBP-Associated Factors - TAFIIs •TAFIIs attach to TBP and extend binding of TFIID beyond TATA box in some promoters • TAFIIs can bind initiator and downstream elements; TAFIIs help initiate transcription from promoters initiators and DPEs •Specifically, TAFII150 and TAFII250 form a ternary complex with TBP and bind to the initiator and DPE -shown by crosslinking and footprinting studies •In certain TATA-less promoters, TAFII250 and TAFII150 bind to initiator or DPE and recruit rest of TFIID to promoter

Drosphila TFIID assembled in vitro

Enhancers and Silencers •Class II promoters have cis-acting DNA elements that influence transcription but are not part of the promoter: enhancers and silencers •Trans-acting factors - proteins - act in conjunction with cis-elements. •Enhancers stimulate transcription while silencers inhibit transcription. Certain elements can act as silencers or enhancers depending on the trans-acting factors present. •Enhancers and silencers are position and orientation- independent.

Transcription activators: DNA binding motif -Zinc containing modules -Homoedomains -bZIP and bHLH domains Activation domain -Acidic -Glutamine rich -Proline rich Diversity of interaction with other transcription factors - semester course in molecular biology on this topic alone

Model for transcriptional activation

Model for initiation, promoter clearance, and elongation

Summary Picture

Eukaryotic transcription termination by RNA Pol II •Mechanism of termination remains unclear. •Involves at least two elements: a polyadenlyation signal and a downstream element (DSE) involved in Pol II pausing.