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The SV40 Promoter

The SV40 Promoter. Enhancer GC boxes (6) TATA box. Transcription Factors for Class II promoters (RNAP II). Basal factors Required for initiation at nearly all promoters; determine site of initiation; interact with TATA box. Upstream factors

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The SV40 Promoter

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  1. The SV40 Promoter Enhancer GC boxes (6) TATA box

  2. Transcription Factors for Class II promoters (RNAP II) • Basal factors Required for initiation at nearly all promoters; determine site of initiation; interact with TATA box. • Upstream factors DNA binding proteins that recognize consensus elements upstream of TATA box. Ubiquitous. Increase efficiency of initiation. Interact with proximal promoter elements (e.g., CCAAT box). • Regulated factors Work like upstream factors but are regulatory. Made or active only at specific times or in specific tissues. Interact with enhancers, silencers, or insulators.

  3. Basal Factors Required to form the Class II Pre-Initiation Complex • 6 factors + RNAP II = Pre-Init. Complex • Factors : • TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH • Many are multi-subunit • Factors + RNAP must also bind in a specific order (in vitro)

  4. Electrophoretic mobility shift assay (EMSA) of TFII factors D,A,B,F on the Adenovirus major late promoter. Conclusions: 1. D and B must form a complex on DNA for Pol II or F to bind. 2. F must bind along with Pol; Pol can’t bind w/o F. Fig. 11.1a

  5. Footprinting TFIID,A,B on a Class II Promoter with phenanthroline–Cu2+ and with DNAse I. D makes footprint, which is enhanced by A. B does not expand the footprint, but makes +10 nt more reactive. Notice that DNase I gives a bigger footprint than OP-Cu2+. Fig. 11.2b

  6. Model for forming the DABPolF complex. (Pol extends the footprint downstream by another 34 bp: total is > 50 bp (-40 to +17). Fig 11.4

  7. TFIID • Binds first • Contains TBP and 8-10 TAFIIs • TBP = TATA box binding protein - 38 kDa protein - Highly conserved C-terminal domain for binding TATA box - Saddle-like structure - Binds DNA via the minor groove

  8. Major Groove has more potential for recognition. D= H-bond donor A= H-bond acceptor Invert and Major Groove still different. Fig 9.14

  9. TBP bends DNA ~80o and forces open the minor groove. Similar to Fig. 11.6

  10. TBP is required by all 3 nRNAPs! 1. A yeast strain with a temperature-sensitive TBP was created. 2. Transcription extracts were prepared from this strain and from wild-type (WT). 3. The extracts were treated at the indicated temperature, and then used to transcribe a gene for each RNAP. 4. Transcription was assayed by S1 mapping the 5’ end of each RNA. rRNA cyc1 Conclusion: the TBP is required by all 3 RNAPs. Fig. 11.7

  11. 2. TAFIIs of TFIID: ~8 mostly conserved proteins ranging from 30  250 kDa 11.9 11.8

  12. Functions of TAFIIs (TFIID): • Strongly promote transcription from promoters with I (initiator) and D (downstream) elements, such as Hsp70 in Drosophila (right). • X-linking (to DNA) and footprinting with different complexes showed that TAFII250 and TAFII150 bind I and D regions in cooperation with TBP. from Fig. 11.10

  13. Footprinting TBP and a Ternary TAF Complex on the HSP70 Promoter Conclusion: The Inr and DPE regions are bound by TAFII250 and/or TAFII150 Fig 11.12

  14. TAFIIs also function to: • Promote transcription from some class II promoters that lack a TATA box. • Interact with some upstream activators (e.g., Sp1), and hence can act as co-activators. • Sp1 interacts with TAFII110 • Gal4 NTF-1 activator works via TAFII150 and TAFII60

  15. TAFs are not universally required. Based mostly on yeast strains with a temperature-sensitive TAFII subunit. RNA from each strain was hybridized to a microarray of 5500 yeast genes.

  16. TBP is also not universally required. • Drosophila has an alternative complex, TRF-1 (TBP-related factor 1), that his its own TAFs (nTAFs) and promotes formation of the pre-initiation complex in neural tissue (binds TFIIA and TFIIB). • The Drosophla tudor gene has two promoters, one with a TATA box that binds TBP, and the other a TC box that binds TRF-1. 11.15

  17. TFIIB (and TFIIA) • TFIIB is needed for the Pol/TFIIF complex to bind to TFIID TFIIB-TBP-Pol II-DNA structure • TFIIA binds to TBP and could be considered a TAFII

  18. TFIIF • 2 subunits, called RAP70 and RAP30 (for RNAP associated protein). • Binds to the RNAP; RAP30 delivers it to the DAB complex. • Reduces non-specific binding of the RNAP to DNA, analogous to the s factor in E. coli.

  19. TFIIE and TFIIH • TFIIE • Binds after Pol/TFIIF binds • Stimulates TFIIH • TFIIH • Also required for promoter clearance • Complex (9 subunits) • DNA helicase activity (for melting DNA) • Kinase activity - phosphorylates the CTD of the large subunit of RNAP Fig 11.20

  20. RNAP is stalled at +10 - +12. TFIIH (+ATP) further unwinds DNA, expanding the bubble and allowing RNAP to go to elongation phase. TEFb further phosphory-lates the CTD. Fig. 11.25

  21. Model for the Class II Preinitiaiton Complex

  22. Elongation Factor TFIIS and Proofreading TFIIS - stimulates elongation by limiting pauses - stimulates proofreading by promoting the RNase activity of the RNAP II 11.28 11.27 11.28

  23. Class I Promoters (for nRNAP I) SL1 UBF -150 -100 -50 +20 UCE Core • SL1 contains TBP and 3 TAFIs • the human complex

  24. Factors for Initiation at Class I Promoters • nRNAP I needs two factors, SL1 and UBF • SL1 binds to the Core element, and provides species specificity - composed of TBP plus 3 TAFIs - the TAFIs are 110, 63, and 48 (different from the TAFs in TFIID) 2. UBF binds UCE (UPE), acts synergistically by promoting SL1 binding to Core

  25. Factors for nRNAP III • Transcription of all class III genes requires TFIIIB and TFIIIC, but TFIIIA needed for transcription of 5S rRNA • TFIIIC - binds the internal promoter • TFIIIB - binds to TFIIIC and upstream DNA, recruits the RNAP • TFIIIA - binds internal promoter, a Zn2+-finger protein

  26. Model for the assembly of the preinitiation complex on a class III tRNA promoter Note: does not need TFIIIA. Fig. 11.39

  27. TFIIIB contains TBP + 2 TAFIIIs In vitro data indicates that TBP is used by all 3 polymerases (TFIID, SL1, TFIIIB). In vivo, the TBP mutant of yeast did not synthesize 45S pre-rRNA, a mRNA, or 5S rRNA.

  28. Pre-initiation complexes on TATA-less promoters for all 3 RNAPs. Assembly factor (green) binds, which promotes binding of a TBP-complex. Fig 11.42

  29. The importance of TBP TBP is important for forming many transcription initiation complexes. It acts by recruiting other proteins, either the RNAP or other general transcription factors (class II). The specificity of TBP for TATA-less promoters – i.e., what kind of promoter it will activate, resides with its TAFs.

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