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 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.

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)

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

D makes footprint, which is enhanced by A. 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

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

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

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

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

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 Top diagram shows the locations of mutations in the conserved C-terminal region of TBP that made it temp. sensitive; red boxes are repeated elements involved in DNA-binding. Conclusion: the TBP is required by all 3 RNAPs. Fig. 11.7

2. TAFIIs of TFIID: ~8 mostly conserved proteins ranging from 30  250 kDa One at right is Drosophila TFIID assembled in vitro with the 8 cloned genes. Model is hypothetical structure. The lines across indicate homologous proteins. 11.9 11.8

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. In vitro transcription with the RNAP plus either TBP or TFIID. With some class II promoters, not much difference between TBP and TFIID –based transcription. from Fig. 11.10

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

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

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.

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

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

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.

TFIIE and TFIIH Fig 11.20 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 Shift in size with ATP only when TFIIH was added (lanes 7 and 8). Fig 11.20

Fig. 11.25 RNAP is stalled at +10 - +12. TBP replaces TFIID, TFIIA not needed, 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

Model for the Class II Preinitiaiton Complex

Elongation Factor TFIIS and Proofreading 11.28 New RNA goes through exit channel, but RNA extruded through pore when the RNAP backtracks or backslides 11.27 TFIIS - stimulates elongation by limiting pauses - stimulates proofreading by promoting the RNase activity of the RNAP II 11.28

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

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

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 Dnase I footprinting of the TFIIIC and B factors binding to a tRNA gene. a) control, b) TFIIIC, c)TFIIIC + TFIIIB, d) like c plus heparin to strip off loosely attached protein

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

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.

Pre-initiation complexes on TATA-less promoters for all 3 RNAPs. Assembly factor (green) binds, which promotes binding of a TBP-complex. Doesn’t show binding of UBF and SL1 to both elements of the class I promoter as some data suggests. Sp1 recruits TFIID to those Class II promoters with GC boxes. Fig 11.42

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.