Molecular biology - Chapter 11: General transcription factors in eukaryotes

Assembly of the preinitiation complex on each kind of eukaryotic promoter begins with binding of assembly factor to promoter TBP is this factor with TATA-containing class II and class III promoters If TBP is not the first bound, it still becomes part of the growing preinitiation complex and serves an organizing function Specificity of TBP depends on associated TAFs

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Molecular Biology Fourth EditionChapter 11General Transcription Factors in EukaryotesLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.111.1 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 transcription start site meltedWhile the class II complex is quite involved, explore it first, then those of classes I and III2The Class II Preinitiation ComplexClass II preinitiation complex contains:Polymerase II6 general transcription factors:TFIIATFIIBTFIIDTFIIETFIIHThe transcription factors (TF) and polymerase bind the preinitiation complex in a specific order3Four Distinct Preinitiation ComplexesTFIID 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 optional4Model of Formation of the DABPolF Complex5Structure 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° curve8 to 10 copies of TBP-associated factors (TAFIIs) specific for class II6The 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 the Archaea, single-celled organisms lacking nuclei7The TBP-Associated FactorsThese are also called TAFIIs8 different proteins are designated by MWMost are evolutionarily conserved in eukaryotesSeveral functions discovered:Interaction with the core promoter elementsInteraction with gene-specific transcription factorsWhen attached to TBP extend the binding of TFIID beyond the TATA box8Model for the Interaction Between TBP and Promoters9Roles of TAFII250 and TAFII150The TAFII250 and TAFII150 help the TFIID bind to the initiator and DPE of promotersAlso aid in TFIID interaction with Sp1 that is bound to GC boxes upstream of the transcription start siteThey enable TBP to bind to:TATA-less promoters that contain elements such as a GC boxTAFII250 has 2 enzymatic activities:Histone acetyltransferaseProtein kinase10Transcription Enhancement by Activators11Exceptions 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 promoters12Structure and Function of TFIIBThe gene for human TFIIB has been cloned and expressed by Reinberg et al.TFIIB binds to TBP at the TATA box via its C-terminal domainPolymerase 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 box13TFIIB 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 complex14TFIIHTFIIH is the last general transcription factor to join the preinitiation complexPlays 2 major roles in transcription initiation:Phosphorylate the CTD of RNA polymerase IIUnwind DNA at the transcription start site to create the transcription bubble15Phosphorylation of the CTD of RNA Polymerase II The preinitiation complex forms with hypophosphorylated form of RNA polymerase IIThen 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 transcription16Phosphorylated Polymerase IIO During ElongationDuring the shift from initiation to elongation, phosphorylation on serine 5 of the heptad repeat is lostIf phosphorylation of serine 2 is also lost, polymerase pauses until rephosphorylation by a non-TFIIH kinase occurs17TFIIHTFIIH is a very complex proteinContains 9 subunitsSeparates into 2 complexesProtein kinase complex of 4 subunitsCore TFIIH complex of 5 subunits with 2 DNA helicase/ATPase activities18Role of TFIIE and TFIIHTFIIE and TFIIH Not essential for Formation of an open promoter complex ElongationRequired for promoter clearance19Participation 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 +1020Expansion 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 promoter21Transcription 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 complex22Schematic Model23The 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 holoenzyme24The Elongation Factor TFIISEukaryotes control transcription primarily at the initiation stepThere is some control exerted at elongationTFIIS, isolated from tumor cells, specifically stimulates transcription 25Elongation and TFIISRNA polymerases do not transcribe at steady rateShort stops in transcription are termed transcription pausesPauses are for variable lengths of timeTend to occur at defined pause sites where DNA sequence at those sites destabilize the RNA-DNA hybrid, causing polymerase to backtrackIf backtracking goes too far, polymerase cannot recover on its own = Transcription arrestPolymerase needs help from TFIIS during a transcription arrest26TFIIS Stimulates Proofreading of TranscriptsTFIIS stimulates proofreading, likely by stimulating RNase activity of the RNA polymeraseThis would allow polymerase to cleave off a misincorporated nucleotide and replace it with a correct oneProofreading is the correction of misincorporated nucleotides2711.2 Class I FactorsRNA polymerase I and 2 transcription factors make up the preinitiation complex, much simpler than the preinitiation complex for class II RNA polymeraseTranscription factors:A core-binding factor, SL1 or TIF-IBA UPE-binding factor, upstream-binding factor (UBF) or upstream activating factor (UAF)28The 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 I29Upstream-Binding FactorThis transcription factor is an assembly factor that helps SL1 to bind to the core promoter elementIt works by bending the DNA dramaticallyDegree of reliance on UBF varies considerably from one organism to anotherSize of polypeptide is 97-kD30Structure and Function of SL1Human SL1 is composed of TBP and TAFs which bind TBP tightly:TAFI110TAFI63 TAFI48These TAFs are completely different from those found in TFIIDYeast and other organisms have TAFIs that are different from the human group3111.3 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 three32TFIIIATFIIIA was the first eukaryotic transcription factor to be discoveredFirst member of the family of DNA-binding proteins that feature a zinc feature to be describedZinc finger is roughly finger-shaped protein domain Contains 4 amino acids that bind a zinc ion33TFIIIB 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 rounds34Scheme for Assembly of Preinitiation ComplexTFIIIC binds to internal promoterTFIIIC promotes binding of TFIIIB with its TFBTFIIIB promotes polymerase III binding at start siteTranscription begins35Model 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 begins36The Role of TBPAssembly of the preinitiation complex on each kind of eukaryotic promoter begins with binding of assembly factor to 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 TAFs37

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