2General pattern of cis-acting control elements that regulate gene expression in yeast and metazoans Whereas control elements for bacterial promoters tend to be located nearby, eukaryotic control elements can be located up to 50 kb upstream or downstream of the gene. Can also be inside the gene.While most Pol II genes have a TATAA box, some don't(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.
3Schematic 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 s 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.Steps in transcriptional initiation.- Recruitment of polymeraseMelting the double helix. Template, non template strandBringing in the first ribonucleotide and making first bondsPolymerization, promoter clearance, bubble movement- Disruption of RNA/DNA helix and re-formation of DNA helix
4Random transcription by bacterial RNA polymerase core E. coli RNA pol core+PromoterRandom initiationelongationTranscripts of random size
5Accurate initiation by bacterial RNA polymerase E. coli RNA polholoenzyme+PromoterinitiationNascent RNAelongationIdentification of Trans-acting factorsOn their own, both bacterial and eukaryotic RNA core polymerases can transcribe RNA, but start sites are not specific. Need other factors to direct polymerase to promoter. For bacterial RNA polymerase, the sigma (σ) factor does this job. For eukaryotic Pol II, need general transcription factors (GTFs)RNA Pol coreRun-off transcript,Discrete size++
6Pol II basic promoter elements Most eukaryotic promoters have TATA box
8Random transcription by eukaryotic RNA polymerase Eukaryotic RNA pol II+PromoterRandom initiationelongationTranscripts of random size
9Fractionation of nuclear extracts to find GTFs Fractionation scheme, DEAE cellulose PlusPurifiedPol IIRun-offTranscriptAccurateInitiation atpromoterNuclear extracts were shown to give accurate initiation. Extracts were then fractioned to look for individual components. Led to identification of TFIID, A, B, F, E , H and Pol II as required for initiation. All components now purified (most are multi-subunit) and genes cloned.Matsui, Segall, Weil, Roeder (1980) JBC 255:11992
10Accurate initiation by Euk RNA polymerase II plus factors in the nucleus Eukaryotic RNA pol II+Nuclear extract, S-100+PromoterGeneralTranscriptionInitiationFactorsinitiationNascent RNAelongationRun-off transcript,Discrete size++
11General transcription factors = GTFs Proteins other than RNA polymerase involved in transcriptionInitiation, Elongation, TerminationGeneral 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
12GTFs for RNA polymerase II Modulates helicaseIIATFIIDTBP}TAFsIIEHelicasehelicaseIIFIIBIIHTargets Pol IIto promoterRecognize core promoterCTD protein kinaseprotein kinaseIIEIIFPol IIaIIBIIAInrIIHTBPCTD of large subunit of Pol IIMany GTFs are possible targets for activators of transcription.
14Sequential Binding Model for assembly of preinitiation complex -30+1TATAInrSequential Binding Model for assembly of preinitiation complexIIBEukaryotic RNApolymerase IITFIID}TBPTAFsIIECTD of large subunit of Pol IIPol IIaor TBPIIAIIFhelicaseprotein kinaseIIHTATAInrpreinitiation complexATP hydrolysisinitiation complex, DNA melted at InrEMSA analysis and commitment assays led to sequencial model for preinitiation complex (PIC) formation: D to DA to DAB PolIIF toDABPolIIFEH.TFIID us multi-subunit complex = TBP + TAFs. TBP components recognizes the TATAA sequence. However, TFIID can also recognize TATAA les promoters by alternate binding modes. TBP is also found in promoter recognition complexes for the other polymerases.= PICPolymerization 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.Activated PIC
15TATA 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.
17TBP bends DNA ~80o and forces open the minor groove.
18Four complexes function as class-specific promoter selectivity factors The association of TBP with TAFIs,TAFIIs, TAFIIIs, 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, 1398.
20Functions of TAFIIs (of TFIID): Strongly promote transcription from promoters with I (initiator) and D (downstream) elements.X-linking (to DNA) and footprinting with different complexes showed that TAFII250 and TAFII150 bind Inr and D regions in cooperation with TBP.
21TAFIIs 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 TAFII110Gal4 NTF-1 activator works via TAFII150 and TAFII60
22TFIIA 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.
23TFIIF2 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 s factorin E. coli.
24TFIIE and TFIIHTFIIEBinds after Pol/TFIIF binds to the pre- initiation complex.Has 2 different subunits, both needed to stimulate transcription.TFIIHRequired for promoter clearanceComplex protein with 9 subunitsHas DNA helicase/ATPase activity (RAD25 gene)for melting DNA at transcription bubbleAlso has Kinase activity: phosphorylates the carboxyl terminal domain (CTD) of the large subunit of RNAP
25TAFs 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.
26RNA Pol II bound to DNA and general transcription initiation factors
27Model for RNA Polymerase II Phosphorylation CTD has repeat of (YSPTSPT)26-50The large subunit of the RNA pol II has a C terminal domain (CTD) containing repeated YSPTSPS. 26 repeats in yeast, 50 in human. Phosphorylation of CTD is important for initiation of transcription and elongation. The form of RNA PolII first isolated could be directed to core promoters by GTFs, but was unresponisve or only minimally to upstream activators.Largest subunit - Rpb1 gene, a.k.a. Subunit II in mice, is phosphorylated on its carboxy- terminal domain (CTD).2 forms of large subunitIIa - non-phosphorylated formIIo - phosphorylated formFunctionally different: IIa-containing enzyme binds promoter; IIo-containing enzyme is in elongation phase.Phosphorylation of Pol IIa to make Pol IIo is needed to release the polymerase from the initiation complex and allow it to start elongation.
28RNAP making short RNAs, its stalled at +10 - +12. TFIIH causes further DNA unwinding, allowing the bubble to grow and RNAP to go to elongation phase.
29Other 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 operonRgr1: resistance to glucose repressionSrb proteinsYeast strains with truncations in the CTD of the large subunit of RNA polymerase B are cold-sensitiveSRB genes: when mutated, suppress the phenotype of CTD deletionsExtragenic suppressors: implicated in RNA polymerase function
30RNA polymerase II Holoenzyme and Mediator RNA polymerase II + (TFIIB, E, F, H )+ (Srb2, 4, 5, 6) + (Rgr1, Gal11, others)Correct initiation in presence of TBP (TFIID)Responds to transcriptional activatorsMediatorComplex needed for a response to transcriptional activators by purified RNA Pol II plus GTFsYeast Mediator has 20 subunits, including Srb2, 4, 5, 6; Srb7, Rgr1, Gal11, Med 1, 2, 6, 7, Pgd1, Nut 1, 2, and othersRNA Pol II + Mediator (+ some GTFs?) = Holoenzyme
32The search for targets of transcription activators: the power of genetics + biochemistryRoger Kornberg: purify “holoenzyme” from nuclear extractslook for a mediator of transcription factor activation in vitro“Mediator” complexKim et al. (1994) Cell 77, 599.Roger Kornberng and coworkers discover the mediator complex by biochemical purification."required reading"
38Enhancers and Silencers Enhancers stimulate transcription, silencers inhibit.Both are orientation independent.Flip 180 degrees, no effectBoth are position independent.Can work at a distance from promoterEnhancers have been found all overBind regulated transcription factors.
39An 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.
40Model for cooperative assembly of an activated transcription-initiation complex at the TTR promoter in hepatocytesFour 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.Basal promoter is usually regulated by additional sequences, simplest of which are upstream elements. In yeast, these are usually within few hundred nucleotides of the TATAA box, but can be much further away in metazoans. Elements can be identified by deletions/mutations.Upstream elements are binding sites for additional DNA binding proteins (transcription factors); simplest in yeast consist of one protein binding site; more complicated upstream elements contain more binding sites.Transcription factors can be simplistically considered to have two functional domains, one to bind DNA and one to activate transcription.Transcription activations domains have been identified by fusing parts of proteins to heterologous DNA binding domains.
41Model of the enhancesome that forms on the b-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.]
42Sp1: Factor for Upstream (Proximal) Class II Promoter Element Binds GC boxes, stimulates transcriptionInteracts with TAFII110 in TFIIDAlso stimulates transcription of TATA-less nRNAP II promoter (by promoting TFIID binding)
43Structure of Eukaryotic Transcription Factors Many have modular structure:DNA-binding domainTranscription activating domainProteins can have > 1 of each, and they can be in different positions in protein.Many also have a dimerization domain
44Recent data suggests SP1 actually has 4 activating domains.
45Required reading:Blau et al., Mol Cell Biol 1996, 16 (5): 204Three functional Classes of Transcriptional Activation Domains
46Activation Domains Acidic (e.g., GAL4, 49 aa domain – 11 acidic aa) Glutamine-rich (e.g., 2 in Sp1, ~25% gln)Proline-rich (e.g., CTF, 84 aa domain – 19 are proline)
47How do different trans-activation domains stimulates transcription How do different trans-activation domains stimulates transcription? INITIATION VS ELONGATION