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Download Transcription in Eukaryotes Eukaryotes have 3 different RNA
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Transcription in Eukaryotes •RNA Polymerases •Promoters •General Transcription Factors 1 Eukaryotes have 3 different RNA Polymerases Figure 10.1 2 1 3 Sensitivity or RNA Pols to α-amanitin Figure 10.4 4 2 Structure of RNA Polymerases •all 3 are large multimeric proteins (500-700 kDa) •all have two large subunits with sequence similarity to β and β’ of E. coli. Therefore, catalytic sites of three RNA Pol I, II and III may be conserved. 5 Epitope tagging to Idetnfiy RNA Pol II subunits Figure 10.6 6 3 Subunit Structure of Yeast RNA Pol II Figure 10.7 7 RNA Polymerase II Structure Summary •Yeast Pol II enzyme consists of 12 different polypeptides (RPB1 - 12). •Core subunits are RPB 1, 2 and 3. •RPB1 and 2 are orthologues of E. coli RNA Pol β and β’. •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 8 4 RNA Polymerase II Structure Summary Continued •RPB 2 (β’ ortholgue) is at or near active site. •RPB3/11 may function as orthologue of α-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 9 Structure of Prokaryotic and Eukaryotic RNA Pols 10 5 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). •Types I and II of Class III promoters have promoter sequence wholly within gene. Type III of Class III promoter resemble Class II promoters. •Class II promoters have up to five conserved elements. 11 First concensus sequence from lining up several eukaryotic promoters: TATA box 12 6 Generic class II promoter Figure 10.20 13 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 orientation14 independent. 7 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. 15 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 16 formation. 8 Model for DABPoF Complex Formation Figure 11.4 17 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. 18 9 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 19 TFIID to promoter Goodrich et al., Cell 84: 825-930, 1996. 20 10