Bài giảng Molecular Biology - Chapter 11 General Transcription Factors in Eukaryotes

Molecular BiologyFifth EditionChapter 11General Transcription Factors in EukaryotesLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.1Transcription in EukaryotesEukaryotic RNA polymerases, unlike their bacterial counterparts, are incapable of binding by themselves to their respective promotersEukaryotic RNA polymerases rely on proteins called transcription factors to show them the wayTwo classes: general transcription factors and gene-specific transcription factors (activators)211.1 Class II FactorsGeneral transcription factors combine with RNA polymerase to form a preinitiation complexThis complex is able to initiate transcription when nucleotides are availableTight binding involves formation of an open promoter complex with DNA at the transcription start site that has melted The assembly of preinitiation complexes involving polymerase II is quite complex3The Class II Preinitiation ComplexClass II preinitiation complex contains:RNA Polymerase II6 general transcription factors:TFIIA, TFIIB, TFIID, TFIIE, and TFIIHThe transcription factors (TF) and polymerase bind the preinitiation complex in a specific order (as studied in vitro)4Four Distinct Preinitiation ComplexesTranscription factors bind to class II promoters in the following order in vitro:TFIID with help from TFIIA binds to the TATA box forming the DA complexTFIIB binds next generating the DAB complexTFIIF helps RNA polymerase bind to a region from -34 to +17, now it is DABPolF complexLast the TFIIE then TFIIH bind to form the complete preinitiation complex = DABPolFEHIn vitro, the participation of TFIIA seems to be optional5Model of Formation of the DABPolF Complex6Structure and Function of TFIIDTFIID contains several subunitsTATA-box binding protein (TBP)Highly evolutionarily conservedBinds to the minor groove of the TATA boxSaddle-shaped TBP lines up with DNAUnderside of the saddle forces open the minor grooveThe TATA box is bent into 80° curveTBP-associated factors (TAFs) specific for class II7Structure of the TBP-TATA box complex8The Versatility of TBPGenetic studies have demonstrated TBP mutant cell extracts are deficient in:Transcription of class II genesTranscription of class I and III genesTBP is a universal transcription factor required by all three classes of genesRequired in transcription of at least some genes of Archaea, single-celled organisms lacking nuclei9The TBP-Associated FactorsThese are also called TAFs (TAFIIs is written to denote transcription of class II genes)13 TAFs have been identified and associated with class II preinitiation complexesThe core TAFs were first named according to their molecular mass but have now been renamed according to their sizes, from largest to smallestSeveral functions discovered:Interaction with the core promoter elementsInteraction with gene-specific transcription factorsWhen attached to TBP extend the binding of TFIID beyond the TATA box10Model for the Interaction Between TBP and Promoters11Roles of TAF1 and TAF2The TAF1 and TAF2 help the TFIID bind to the initiator and DPE of promotersThey enable TBP to bind to TATA-less promoters that contain elements such as a GC boxDifferent combinations of TAFs are required to respond to variosu activators, at least in higher eukaryotesTAF1 has two enzymatic activities:Histone acetyltransferase (HAT)Protein kinase12Transcription Enhancement by Activators13Exceptions to the Universality of TAFs and TBPTAFs are not universally required for transcription of class II genesEven TBP is not universally requiredSome promoters in higher eukaryotes respond to an alternative protein such as TRF1 (TBP-related factor 1)The general transcription factor NC2: Stimulates transcription from DPE-containing promotersRepresses transcription from TATA-containing promoters14Structure and Function of TFIIBStructural studies have revealed that TFIIB binds to TBP at the TATA box via its C-terminal domain and polymerase II via its N-terminal domainThe protein provides a bridging action that effects a coarse positioning of polymerase active center about 25 –30 bp downstream of the TATA boxPlays an important role in establishing the transcription start site15TFIIB DomainsA loop motif of the N-terminal domain in TFIIB effects a fine positioning of the transcription start by interacting with template ssDNA near the active centerTFIIB N-terminal domain, finger and linker domains, lies close to the RNA polymerase II active center and to largest subunit of TFIIF in preinitiation complex16TFIIHTFIIH is the last general transcription factor to join the preinitiation complex (contains 9 subunits)Separates into 2 complexesProtein kinase complex of 4 subunitsCore TFIIH complex of 5 subunits with 2 DNA helicase/ATPase activitiesPlays two major roles in transcription initiation:Phosphorylates the CTD of RNA polymerase IIUnwinds DNA at the transcription start site to create the “transcription bubble”17Phosphorylation of the CTD of RNA Polymerase II The preinitiation complex forms with hypophosphorylated form of RNA polymerase II (IIA)Then TFIIH phosphorylates serines 2 and 5 in the heptad repeat in the carboxyl-terminal domain (CTD) of the largest RNA polymerase subunitThis creates the phosphorylated form of the polymerase enzyme (IIO)This phosphorylation is essential for initiation of transcription18Phosphorylated Polymerase IIO During ElongationDuring the shift from initiation to elongation, two serines of the CTD are phosphorylated (serines 2 and 5 - and sometimes serine 7)Evidence exists that transcription complexes near the promoter have CTDs in which serine 5 is phosphorylated but that this phosphorylation shifts to serine 2 as transcription progressesTFIIH phosphorylates serine 5 and CTDK-1 (in yeast) phosphorylates serine 219Role of TFIIE and TFIIHTFIIE and TFIIH are not essential for the formation of an open promoter complex or for elongationRequired for promoter clearanceTFIIH has DNA helicase activity that is essential for transcription, presumably because it causes full melting of the DNA at the promoter and thereby facilitates promoter clearance20Participation of General Transcription Factors in InitiationTFIID with TFIIB, TFIIF and RNA polymerase II form a minimal initiation complex at the initiatorAddition of TFIIH, TFIIE and ATP allow DNA melting at the initiator region and partial phosphorylation of the CTD of largest RNA polymerase subunitThese events allow production of abortive transcripts as the transcription stalls at about +1021Expansion of the Transcription BubbleEnergy is provided by ATPDNA helicase of TFIIH causes unwinding of the DNAExpansion of the transcription bubble releases the stalled polymerasePolymerase is now able to clear the promoter22Transcription Factors in ElongationElongation complex continues elongating the RNA when: Polymerase CTD is further phosphorylated by TEFbNTPs are continuously availableTBP and TFIIB remain at the promoterTFIIE and TFIIH are not needed for elongation and dissociate from the elongation complex23Model for the participation of GTFs in initiation, promoter clearance, and elongation24The Mediator Complex and the RNA Polymerase II HoloenzymeMediator is a collection of proteins also considered to be a general transcription factor as it is a part of most class II preinitiation complexesMediator is not required for initiation, but it is required for activated transcriptionIt is possible to assemble the preinitiation complex adding general transcription factors to RNA polymerase II holoenzyme25Eukaryotic Control of TranscriptionEukaryotes control transcription primarily at the initiation stepThere is also some control exerted during elongation, which can involve overcoming transcription pausing or transcription arrestRNA polymerases do not transcribe at a steady rate as they pause, sometimes for a long time, before resuming transcriptionTend to pause at pause sites or DNA sequences that destabilize the DNA-RNA hybrid and cause the polymerase to backtrack26Promoter Proximal PausingA sizable fraction of genes contain specific pause sites lying 20-50bp downstream of the transcription start siteTwo protein factors are known to help stabilize RNA polymerase II in the paused state - DRB sensitivity inducing factor (DSIF) and negative elongation factor (NELF)The signal to leave the paused state is delivered by the positive elongation factor-b (P-TEFb), which is a protein kinase that can phosphorylate polymerase II, DSIF, and NELF27TFIIS Stimulates Proofreading of TranscriptsTFIIS stimulates proofreading, the correction of misincorporated nucleotides, likely by stimulating RNase activity of the RNA polymeraseThis would allow polymerase to cleave off a misincorporated nucleotide and replace it with a correct one2811.2 Class I FactorsThe preinitiation complex that forms at rRNA promoters is much simpler than the preinitiation complex for class II RNA polymeraseIt involves polymerase 1 plus two additional transcription factors:A core-binding factor, SL1 or TIF-IBA UPE-binding factor, upstream-binding factor (UBF) or upstream activating factor (UAF)29The Core-Binding FactorThe core-binding factor, SL1, was originally isolated on the basis of its ability to direct polymerase initiation SL1 also shows species specificityThis factor is the fundamental transcription factor required to recruit RNA polymerase I30Upstream-Binding Factor (UBF)This transcription factor is an assembly factor that helps the core binding factor to bind to the core promoter elementIt works by bending the DNA dramaticallyDegree of reliance on UBF varies considerably from one organism to anotherHuman UBF is a transcription factor that stimulates transcription by polymerase I and can activate the intact promoter, or the core element alone, and it mediates the activation by the UPE31Structure and Function of SL1Human SL1 is composed of TBP and three TAFs (TAFI110, TAFI63, TAFI48) which bind TBP tightlyThese TAFs are completely different from those found in TFIIDYeast and other organisms have TAFIs that are different from the human group3211.3 Class III FactorsIn 1980 a transcription factor was found that bound to the internal promoter of the 5S rRNA gene and stimulated its transcription – TFIIIA Two other transcription factors TFIIIB and TFIIIC have also been studiedTranscription of all classical class III genes requires TFIIIB and TFIIICTranscription of 5S rRNA genes requires all three33TFIIIATFIIIA was the first eukaryotic transcription factor to be discoveredFirst member of the family of DNA-binding proteins that feature a zinc finger to be describedZinc finger is roughly finger-shaped protein domain that contains 4 amino acids that bind a zinc ionHas nine zinc fingers that appear to insert into the major groove on either side of the promoter for the 5S rRNA gene34TFIIIB and TFIIICBoth of these transcription factors are required for transcription of the classical polymerase III genesThey depend on each other for their activitiesTFIIIC is an assembly factor that allows TFIIIB to bind to the region just upstream of the transcription start siteTFIIIB can remain bound and sponsor initiation of repeated transcription rounds35Scheme for Assembly of the Preinitiation Complex on a classical class II promoterTFIIIC binds to internal promoterTFIIIC promotes binding of TFIIIB with its TBPTFIIIB promotes polymerase III binding at start siteTranscription begins36Model of Preinitiation Complex on TATA-Less PromoterAssembly factor binds firstAnother factor, containing TBP, is now attractedComplex now sufficient to recruit polymerase except for class II Transcription begins37The Role of TBPAssembly of the preinitiation complex on each kind of eukaryotic promoter begins with binding of an assembly factor to the promoterTBP is this factor with TATA-containing class II and class III promotersIf TBP is not the first bound, it still becomes part of the growing preinitiation complex and serves an organizing functionSpecificity of TBP depends on associated TAFs38