BIOL 367 Chapter 11: biol 367 chapter 11

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CHAPTER 11 – GENERAL TRANSCRIPTION FACTORS IN EUKARYOTES CLASS 2 FACTORS - General transcription factors combine with RNA polymerase to form a preinitiation complex - Involves formation of an open promoter complex in which the DNA at the transcription start site has melted to allow the polymerase to read it The Class II Preinitiation Complex - contains polymerase II and 6 general transcription factors named TFFIA, TFIIB,TFIID, TFIIE, TFIIF, and TFIIH - They bind in a specific order to the growing preinitiation complex, at least in vitro - This experiment reveals the existence of 4 distinct complexes, which are labeled at the left of the figure. - When the investigators added TFIID and A alone to DNA containing the adenovirus major late promoter, a DA complex formed (lane 1) - When they added TFIIB in addition to D and A, DAB complex formed (lane 2) - Central part of figure shows when the yadded various concentrations of RNA polymerase II andTFIIF to the DAB complex. - In lane 3, all4 factorswere present, but no RNA polymerase  no difference was detectable between the complex formed with these four factors and the DAB complex. Thus, TFIIF does not seem to bind independently to DAB - When the investigators added increasing amounts of polymerase (lanes 4-7), two new complexes appeared - Maximum amount of DABPolF,then started decreasing amount of TFIIF (lanes 8-11). This decreased the yield of DABPolF, ubtil, with no TFIIF but plenty of polymerase (lane 12), essentially no DABPolFcomplexes formed  indicates that RNA polymerase and TFIIFare needed together to join the growing preinitiation complex - Lane 13 left out TFIID  no complexes formed. TFIID is the first factor to bind; the binding of all the other factors dpends on the prsence of TFIID at the TATA box - Lane 14 – no TFIIB  shows that TFIIB was needed to add polymerase and TFIIF - Lane 15 shows that leaving out TFIIA made little difference 1 - they could start with the DBPolF complex and then add TFIIE and TFIIH in turn, producing a larger complex, with reduced mobility, with each added factor - The order of addition of the general transcription factors (and RNA pol) to the preinitiation complex in vitro is as follows: TFIID (or TFIIA+TFIID), TFIIB, TFIIF + polymerase II, TFIIE, TFIIH - TFIID and A protect the TATA box - Whereas TFIID, A and B protected the TATA box region (between -17 and -42) in the DAB complex, RNA pol II and TFIIF extended this protected region another 34 bases on the nontemplate strand (-17 to +17) - Footprinting the DA and DAB complexes - Both the DA and DAB complexes were centered on the TATA box Footprinting of the DABPolF  - When RNA polymerase and TFIIF joined the complex, they caused a large extension of the footprint, to about position +17 – this is consistent with the large size of RNA pol II 2 ← Model for formation of he DABPolF -TFIIF (green) binds to polymerase II (red) and together they join the DAB complex. -Polymerase II extends the DAB footprint in the downstream dierction, and therefore binds ot the DNA downstream of the binding sites for TFIID, A and B, which center on the TATA box Structure of Function of TFIID -Containing a TATA-box-binding protein (TBP) and 13 core TBP- associated factors The TATA-Box-Binding Protein -The TBP in TFIID binds to theminor groove of the TATA box -Changed all the bases of the TATA box, such that the majo rgroove was changed, but the minor groove was not -Made an adenovirus major late TATA box with all C’s instead of T’s, and al lthe I’s instead of A’s (CICIIII instead of TATAAAA) (b) -The CICI box worked just as well as the TATA box, but a nonspecific DNA did not bind TFIID at all. Therefore changing the bases in the TATA box did not affect TFIID binding as long as the minor grove was unaltered - TBP sits on DNA the way as a saddle sits on a horse - The curved undersurface of the saddle, instead of fitting neatly over the DNA, is roughly alilgned wit hthe long axis of the DNA, so its curvature forces the DNA to bend through an angle of 80 degrees  the minor groove is forced open - Two phenylalanine side chains from the stirrups of TBP intercalate, or insert, between base pairs, causing the DNA to kink - Take home: TBP and therefore TFIID binds to the minor groove at TATAA elements The Versatility of TBP - This factor functions not only with poylermase II promoters that have a TATA box, but with TATA-less polymerase II - It also functions with TATA-less polymerase III promoters, and with TATA-less polymerase I promoters - It also seems to be involved in transcription in a whole different kingdom of organisms: the archae 3 The TBP-associated Factors - Used an antibody specific for TBP to immunoprecipiate TFIID from a crude TFIID preparation. Then they treated the immunoprecipitate with 2.5 M urea to strip the TAFs off of the TBP- antibody precipitate and displayed the TAFs by SDS- PAGE  led to identification of 13 TAFs associated with class II preinitiation complexes - TAFs apparently help TBP facilitate transcription from promoters with initiators and DPEs - Which Tafs are responsible for recognizing the initiation and DPE?  TAF1 and TAF2 cooperate in binding to the initiator alone, as well as to the initiator plus a DPE - Promoters that lack a TATA box? Even though these promoters cannot bind TBP directly, most still depend on this transcription factor for activity - TATA-less promoters constian other elements that ensure the binding of TBP. Thsee other elements can be initiators and DPEs, to which TAF1 and TAF2 can bind and thereby secure the whole TFIID to the promoter - Or they can be upstream elements that bind gene-specific transcription factors, which in turn interact with one or more TAFs to anchor TFIId to the promoter - The second major activity of the TAFs is to participate in the transcription stimulation provided by activators - TFIID is sufficient to participate in such stimulation by the factor Sp1, but TBP is not - Different activators work with different combinations of TAFs to enhance transcription, and all of them seem ot have TAF1 in common. This suggests that TAF1 serves as an assembly factor around which other TAFs can aggregate 4 a) TATA containing promoter. TBP can bind by itself to the TATA box of this promoter. It can also bind in the company of all the TAFs in TFIID. And it can bind with a subset of TAFs (bottom) b) TATA-less promoter with initiatior element and DPE. TBP cannot bind by itself to the promoter. The whole TFIID is competent to bind to the TATA-less promoter through interactions between TAF1 (yellow) and TAF2 (brown). TAF1 and TAF2 are sufficient to tether TBP to the initiator and DPE c) TATA less promoter with GC boxes. TBP cannot bind to promoter by itself. The whole TFIID can bind to this promoter through itneractions with Sp1 bound at the GC boxes (middle). TAF1, TAF2, and TAF4 are sufficient ot anchor tBP to the Sp1 bound to the GC boxes - TAFs can have enzymatic activities - TFIIA is essential for TBP (or TFIID) binding to promoters - Mutations in either of the genes encoding the two subunits of TFIIA in yeast are lethal - TFIIA not only stabilizes TBP-TATA box binding, it also stimulates TFIID-promoter binding by an antireprsesion mechanism as follows: When TFIID is not bound to a promoter, the DNA-binding surface of TBP is covered by the N terminal domain of TAF1, which inhibits TFIID binding ot the promoter. But TFIIA can interfere with the interaction ebtween the TAF1 N-terminal domain and the DNA-binding surface of TBP, freeing up TBP for binding to the promoter 5 - It is striking that only 16% of the yeast genes analyzed were as dependent on TAF1 as they were on Rpb1, indicating that TAF1 is required for transcription of only 16% of yeast genes. This is not what we would expect if the TAFs are essential parts of TFIID, and TFIID is an essential part of the preinitiation complexes formed at allclass II genes. - TAF1 and its homolog in higher organisms appeaer to be required in the preinitiation complexes formed at only a subset of genes. Inyeast, these genes tend to be ones governing progression through the cell cycle - TBP is not universally found in preinitiation complexes in higher eukaryotes. The most celebrated exmape of an alternative TBP is TRF1 (TBP-related factor 1) in drosophila. This protein is expressed in developing neural tissue, binds to TFIIA and TFIIB, and stiulates transcription just as TBP does, and it has its own group of TRF-associated factors called nTAFs - TRF appears to be a cell type-specific variant of TBP - Another TBP-like factor (TLF) has been found in all multicellular animals investigated to date. TLF differs from TBP in lacking the pairs of phenylalanines that intercalate between base pairs in tATA boxes and helpbend DNA at the promoter. - TLF appears not ot bind to TATA boxes and may direct transcription at other, TATA-less promoters - TBP-free TAF-containing complex (TFTC) that is able to sponsor preinitiation complex formation without any help from TFIID or TBP 6 Structure and Function of TFIIB - TFIIB is a single-subunit factor - It is the third general transcription factor to join the preinitiation complex in vitro(After TFIID and A), or the second if TFIIA has not yet bound - TFIIB is part of the measuring device that palces RNA pol II in the proper position to initiate transcrption. If so – TFIIB should have two domains: one to bind to each of these proteins - TFIIB does have 2domains: an N-terminal domain and a C terminal domain. These two domains really do function to bridge between TFIID at the TATA box and RNA pol II so as to position the active center of the polymerase about 26-31 bp downstream of the TATA box, just where transcription should begin - TBP,by bending the DNA at the TATA box, wraps the DNA around TFIIB , and thaC TFIIB binds to N a site on the polymerase that positions the enzyme correctly at the transcription