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General pattern of cis-acting control elements that regulate gene expression in yeast and metazoans (a) Genes of multicellular organisms contain both.

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Presentation on theme: "General pattern of cis-acting control elements that regulate gene expression in yeast and metazoans (a) Genes of multicellular organisms contain both."— Presentation transcript:

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2 General pattern of cis-acting control elements that regulate gene expression in yeast and metazoans (a) Genes of multicellular organisms contain both promoter-proximal elements and enhancers as well as a TATA box or other promoter element. The latter positions RNA polymerase II to initiate transcription at the start site and influences the rate of transcription. Enhancers may be either upstream or downstream and as far away as 50 kb from the transcription start site. In some cases, promoter-proximal elements occur downstream from the start site as well. (b) Most yeast genes contain only one regulatory region, called an upstream activating sequence (UAS), and a TATA box, which is 90 base pairs upstream from the start site.

3 Schematic diagram of the steps in the initiation of RNA synthesis (DNA transcription) catalyzed by RNA polymerase. The enzyme first forms a closed complex in which the two DNA strands remain fully base- paired. In the next step the enzyme catalyzes the opening of a little more than one turn of the DNA helix to form an open complex, in which the template DNA strand is exposed for the initiation of an RNA chain. The polymerase containing the bound subunit, however, behaves as though it is tethered to the promoter site: it seems unable to proceed with the elongation of the RNA chain and on its own frequently synthesizes and releases short RNA chains. As indicated, the conversion to an actively elongating polymerase requires the release of initiation factors (the sigma subunit in the case of the E. coli enzyme) and generally involves the binding of other proteins that serve as elongation factors.

4 Random transcription by bacterial RNA polymerase core Promoter E. coli RNA pol core + Random initiation elongation Transcripts of random size

5 Accurate initiation by bacterial RNA polymerase Promoter E. coli RNA pol holoenzyme + initiation elongation Nascent RNA Run-off transcript, Discrete size ++ RNA Pol core

6 Pol II basic promoter elements

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8 Random transcription by eukaryotic RNA polymerase Promoter Eukaryotic RNA pol II + Random initiation elongation Transcripts of random size

9 Fractionation of nuclear extracts to find GTFs Fractionation scheme, DEAE cellulose Matsui, Segall, Weil, Roeder (1980) JBC 255:11992 Plus Purified Pol II Run-off Transcript Accurate Initiation at promoter

10 Accurate initiation by Euk RNA polymerase II plus factors in the nucleus Promoter + initiation elongation Nascent RNA Run-off transcript, Discrete size ++ Eukaryotic RNA pol II + Nuclear extract, S-100 General Transcription Initiation Factors

11 General transcription factors = GTFs Proteins other than RNA polymerase involved in transcription –Initiation, Elongation, Termination General transcription initiation factors (GTIFs) –Proteins required for specific transcription from a minimal promoter (core) –Not subunits of purified RNA polymerase. –Required for RNA polymerase to bind avidly and specifically to promoters. –GTIFs for RNA polymerase II are called TFIIx, where x = A, B, D, … –Can have multiple subunits

12 GTFs for RNA polymerase II TFIID TBP } TAFs IIB IIA IIE IIF IIH helicase protein kinase TBP Inr IIB IIA Pol IIa IIF IIE IIH CTD of large subunit of Pol II Recognize core promoter Targets Pol II to promoter Modulates helicase Helicase CTD protein kinase Many GTFs are possible targets for activators of transcription.

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14 Sequential Binding Model for assembly of preinitiation complex TATA Inr Polymerization of 1st few NTPs and phosphorylation of CTD leads to promoter clearance. TFIIB, TFIIE and TFIIH dissociate, PolII+IIF elongates, and TFIID + TFIIA stays at TATA. IIB Eukaryotic RNA polymerase II TFIID } TBP TAFs IIB IIE CTD of large subunit of Pol II Pol IIa or TBP IIA IIF helicase protein kinase IIH TATA Inr IIA Pol IIa IIF IIE preinitiation complex TATA Inr IIA IIB Pol IIa IIF IIE ATP hydrolysis initiation complex, DNA melted at Inr IIH = PIC Activated PIC

15 TATA Binding Protein = TBP TBP binds in the narrow groove of DNA at the TATA box found about bp 5 to the start site for transcription of many (but not all) genes transcribed by RNA polymerase II. TBP bends the DNA about 90 degrees. TBP alone or with TBP-associated proteins (TAFs) plays an important role in recognizing the core promoter and recruiting RNA polymerase II to the promoter.

16 The Structure of 28kD TBP and TBP/DNA

17 TBP bends DNA ~80 o and forces open the minor groove.

18 The association of TBP with TAF I s,TAF II s, TAF III s, and PTF/SNAPc directs TBP to different promoter classes. The distribution of TBP among these factors contributes to the global regulation of gene expression. From Lee and Young (1998) Regulation of gene expression by TBP-associated proteins. Genes Dev. 12, Four complexes function as class-specific promoter selectivity factors

19 TAFIIs of TFIID: -8 mostly conserved proteins ranging from kDa

20 1.Strongly promote transcription from promoters with I (initiator) and D (downstream) elements. 2.X-linking (to DNA) and footprinting with different complexes showed that TAF II 250 and TAF II 150 bind Inr and D regions in cooperation with TBP. Functions of TAFIIs (of TFIID):

21 TAFIIs also function to: 1.Promote transcription from some class II promoters that lack a TATA box. 2.Interact with some upstream activators (e.g., Sp1), and hence can act as co- activators. Sp1 interacts with TAF II 110 Gal4 NTF-1 activator works via TAF II 150 and TAF II 60

22 TFIIA and TFIIB TFIIA binds to TBP and could be considered a TAFII TFIIB is needed for the Pol/TFIIF complex to bind to TFIID, and can be though of as a linker between these two. A current model has TFIIA binding TBP on the upstream side, with TFIIB binding on the downstream side.

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

24 TFIIE and TFIIH TFIIE 1.Binds after Pol/TFIIF binds to the pre-initiation complex. 2.Has 2 different subunits, both needed to stimulate transcription. TFIIH 1.Required for promoter clearance 2.Complex protein with 9 subunits 3.Has DNA helicase/ATPase activity (RAD25 gene) for melting DNA at transcription bubble 4.Also has Kinase activity: phosphorylates the carboxyl terminal domain (CTD) of the large subunit of RNAP

25 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.

26 RNA Pol II bound to DNA and general transcription initiation factors

27 Model for RNA Polymerase II Phosphorylation Phosphorylation of Pol IIa to make Pol IIo is needed to release the polymerase from the initiation complex and allow it to start elongation. CTD has repeat of (YSPTSPT) 26-50

28 RNAP making short RNAs, its stalled at TFIIH causes further DNA unwinding, allowing the bubble to grow and RNAP to go to elongation phase.

29 Other proteins involved in transcription and regulation In addition to RNA polymerase II and GTFs: Proteins required for regulation, e.g. –Gal11: regulation of the GAL operon –Rgr1: resistance to glucose repression Srb proteins –Yeast strains with truncations in the CTD of the large subunit of RNA polymerase B are cold-sensitive –SRB genes: when mutated, suppress the phenotype of CTD deletions –Extragenic suppressors: implicated in RNA polymerase function

30 RNA polymerase II Holoenzyme and Mediator Holoenzyme –RNA polymerase II + (TFIIB, E, F, H )+ (Srb2, 4, 5, 6) + (Rgr1, Gal11, others) –Correct initiation in presence of TBP (TFIID) –Responds to transcriptional activators Mediator –Complex needed for a response to transcriptional activators by purified RNA Pol II plus GTFs –Yeast Mediator has 20 subunits, including Srb2, 4, 5, 6; Srb7, Rgr1, Gal11, Med 1, 2, 6, 7, Pgd1, Nut 1, 2, and others RNA Pol II + Mediator (+ some GTFs?) = Holoenzyme

31 Expanding the functions of RNA polymerase

32 The search for targets of transcription activators: the power of genetics + biochemistry Roger Kornberg: purify holoenzyme from nuclear extracts look for a mediator of transcription factor activation in vitro Mediator complex Kim et al. (1994) Cell 77, 599. "required reading"

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34 Stages in Initiation of Transcription Bacterial transcription Closed complex: holoenzyme+promoter Open complex (DNA melting, not need ATP ) Abortive transcripton Productive initiation –Transcribe past +9 –Sigma dissociates Elongation Eukaryotic transcription Preinitiation complex (PIC) assembly PIC activation (DNA melting, needs ATP) Abortive transcription Productive initiation –CTD phosphorylated –Promoter clearance Elongation

35 Parallels between initiation pathway in prokaryotes and eukaryotes From Eick et al. (1994) Trends in Genetics 10:

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38 Enhancers and Silencers Enhancers stimulate transcription, silencers inhibit. Both are orientation independent. –Flip 180 degrees, no effect Both are position independent. –Can work at a distance from promoter –Enhancers have been found all over Bind regulated transcription factors.

39 An enhancer in an intron of a gamma-globulin gene. (a) Genes were constructed with the enhancer inverted (B), with it moved upstream of the gene (C) and inverted (D). The DNAs were transfected into mouse cells and synthesis of the protein was assessed by pulse-labeling with a radioactive amino acid, immunoprecipitation, and separation by SDS-PAGE and autoradiography.

40 Four activators enriched in hepatocytes plus the ubiquitous AP1 factor bind to sites in the hepatocytespecific enhancer and promoter-proximal region of the TTR gene. The activation domains of the bound activators interact extensively with co-activators, TAF subunits of TFIID, Srb/Mediator proteins, and general transcription factors, resulting in looping of the DNA and formation of a stable activated initiation complex. Because of the highly cooperative nature of complex assembly, an initiation complex does not form on the TTR promoter in intestinal epithelial cells, which contain only two of the four hepatocyte-enriched transcription factors. Many of the general transcription factors, Srb/Mediator proteins, and RNA polymerase II (Pol II) may be pre- assembled into a holoenzyme complex. Model for cooperative assembly of an activated transcription- initiation complex at the TTR promoter in hepatocytes

41 Model of the enhancesome that forms on the - interferon enhancer. Heterodimeric cJun/ATF-2, IRF-3, IRF-7, and NF-KB (a heterodimer of p50 and p65) bind to the four control elements in the 70-bp enhancer. Cooperative binding of these transcription factors is facilitated by HMGI, which binds to the minor groove of DNA. The cJun, ATF-2, p50, and p65 proteins all appear to interact directly with an HMGI bound adjacent to them. Bending of the enhancer sequence resulting from HMGI binding is critical to formation of an enhancesome. Different DNA-bending proteins act similarly at other enhancers. [Adapted from D. Thanos and T. Maniatis, 1995, Cell 83:1091 and M. A. Wathel et al., 1998,Mol. Cell 1:507.]

42 Sp1: Factor for Upstream (Proximal) Class II Promoter Element Binds GC boxes, stimulates transcription Interacts with TAF II 110 in TFIID Also stimulates transcription of TATA- less nRNAP II promoter (by promoting TFIID binding)

43 Structure of Eukaryotic Transcription Factors Many have modular structure: 1.DNA-binding domain 2.Transcription activating domain Proteins can have > 1 of each, and they can be in different positions in protein. Many also have a dimerization domain

44 Recent data suggests SP1 actually has 4 activating domains.

45 Blau et al., Mol Cell Biol 1996, 16 (5): 204 Three functional Classes of Transcriptional Activation Domains Required reading:

46 Activation Domains 1.Acidic (e.g., GAL4, 49 aa domain – 11 acidic aa) 2.Glutamine-rich (e.g., 2 in Sp1, ~25% gln) 3.Proline-rich (e.g., CTF, 84 aa domain – 19 are proline)

47 How do different trans-activation domains stimulates transcription? INITIATION VS ELONGATION

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53 Three Functional classes of activation domains:


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