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Lecture PowerPoint to accompany Molecular Biology Fifth Edition Robert F. Weaver Chapter 11 General Transcription Factors in Eukaryotes Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Transcription in Eukaryotes • Eukaryotic RNA polymerases, unlike their bacterial counterparts, are incapable of binding by themselves to their respective promoters • Eukaryotic RNA polymerases rely on proteins called transcription factors to show them the way • Two classes: general transcription factors and gene-specific transcription factors (activators) 11-2 11.1 Class II Factors • General transcription factors combine with RNA polymerase to form a preinitiation complex – This complex is able to initiate transcription when nucleotides are available – Tight 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 complex 11-3 The Class II Preinitiation Complex • Class II preinitiation complex contains: – RNA Polymerase II – 6 general transcription factors: • TFIIA, TFIIB, TFIID, TFIIE, and TFIIH • The transcription factors (TF) and polymerase bind the preinitiation complex in a specific order (as studied in vitro) 11-4 Four Distinct Preinitiation Complexes • Transcription 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 complex • TFIIB binds next generating the DAB complex • TFIIF helps RNA polymerase bind to a region from -34 to +17, now it is DABPolF complex • Last the TFIIE then TFIIH bind to form the complete preinitiation complex = DABPolFEH • In vitro, the participation of TFIIA seems to be optional 11-5 Model of Formation of the DABPolF Complex 11-6 Structure and Function of TFIID TFIID contains several subunits – TATA-box binding protein (TBP) • Highly evolutionarily conserved • Binds to the minor groove of the TATA box – Saddle-shaped TBP lines up with DNA – Underside of the saddle forces open the minor groove – The TATA box is bent into 80° curve – TBP-associated factors (TAFs) specific for class II 11-7 Structure of the TBP-TATA box complex 11-8 The Versatility of TBP • Genetic studies have demonstrated TBP mutant cell extracts are deficient in: – Transcription of class II genes – Transcription of class I and III genes • TBP is a universal transcription factor required by all three classes of genes • Required in transcription of at least some genes of Archaea, single-celled organisms lacking nuclei 11-9 The TBP-Associated Factors • These 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 complexes • The core TAFs were first named according to their molecular mass but have now been renamed according to their sizes, from largest to smallest • Several functions discovered: – Interaction with the core promoter elements – Interaction with gene-specific transcription factors – When attached to TBP extend the binding of TFIID beyond the TATA box 11-10 Model for the Interaction Between TBP and Promoters 11-11 Roles of TAF1 and TAF2 • The TAF1 and TAF2 help the TFIID bind to the initiator and DPE of promoters • They enable TBP to bind to TATA-less promoters that contain elements such as a GC box • Different combinations of TAFs are required to respond to variosu activators, at least in higher eukaryotes • TAF1 has two enzymatic activities: – Histone acetyltransferase (HAT) – Protein kinase 11-12 Transcription Enhancement by Activators 11-13 Exceptions to the Universality of TAFs and TBP • TAFs are not universally required for transcription of class II genes • Even TBP is not universally required • Some promoters in higher eukaryotes respond to an alternative protein such as TRF1 (TBPrelated factor 1) • The general transcription factor NC2: – Stimulates transcription from DPE-containing promoters – Represses transcription from TATA-containing promoters 11-14 Structure and Function of TFIIB • Structural studies have revealed that TFIIB binds to TBP at the TATA box via its Cterminal domain and polymerase II via its N-terminal domain • The protein provides a bridging action that effects a coarse positioning of polymerase active center about 25 –30 bp downstream of the TATA box • Plays an important role in establishing the transcription start site 11-15 TFIIB Domains • A 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 center • TFIIB N-terminal domain, finger and linker domains, lies close to the RNA polymerase II active center and to largest subunit of TFIIF in preinitiation complex 11-16 TFIIH • TFIIH is the last general transcription factor to join the preinitiation complex (contains 9 subunits) • Separates into 2 complexes • Protein kinase complex of 4 subunits • Core TFIIH complex of 5 subunits with 2 DNA helicase/ATPase activities • Plays two major roles in transcription initiation: – Phosphorylates the CTD of RNA polymerase II – Unwinds DNA at the transcription start site to create the “transcription bubble” 11-17 Phosphorylation 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 carboxylterminal domain (CTD) of the largest RNA polymerase subunit – This creates the phosphorylated form of the polymerase enzyme (IIO) – This phosphorylation is essential for initiation of transcription 11-18 Phosphorylated Polymerase IIO During Elongation • During 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 progresses • TFIIH phosphorylates serine 5 and CTDK-1 (in yeast) phosphorylates serine 2 11-19 Role of TFIIE and TFIIH TFIIE and TFIIH are not essential for the formation of an open promoter complex or for elongation • Required for promoter clearance • TFIIH 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 clearance 11-20 Participation of General Transcription Factors in Initiation • TFIID with TFIIB, TFIIF and RNA polymerase II form a minimal initiation complex at the initiator • Addition of TFIIH, TFIIE and ATP allow DNA melting at the initiator region and partial phosphorylation of the CTD of largest RNA polymerase subunit • These events allow production of abortive transcripts as the transcription stalls at about +10 11-21 Expansion of the Transcription Bubble • Energy is provided by ATP • DNA helicase of TFIIH causes unwinding of the DNA • Expansion of the transcription bubble releases the stalled polymerase • Polymerase is now able to clear the promoter 11-22 Transcription Factors in Elongation • Elongation complex continues elongating the RNA when: – Polymerase CTD is further phosphorylated by TEFb – NTPs are continuously available • TBP and TFIIB remain at the promoter • TFIIE and TFIIH are not needed for elongation and dissociate from the elongation complex 11-23 Model for the participation of GTFs in initiation, promoter clearance, and elongation 11-24 The Mediator Complex and the RNA Polymerase II Holoenzyme • Mediator is a collection of proteins also considered to be a general transcription factor as it is a part of most class II preinitiation complexes • Mediator is not required for initiation, but it is required for activated transcription • It is possible to assemble the preinitiation complex adding general transcription factors to RNA polymerase II holoenzyme 11-25 Eukaryotic Control of Transcription • Eukaryotes control transcription primarily at the initiation step • There is also some control exerted during elongation, which can involve overcoming transcription pausing or transcription arrest • RNA polymerases do not transcribe at a steady rate as they pause, sometimes for a long time, before resuming transcription • Tend to pause at pause sites or DNA sequences that destabilize the DNA-RNA hybrid and cause the polymerase to backtrack 11-26 Promoter Proximal Pausing • A sizable fraction of genes contain specific pause sites lying 20-50bp downstream of the transcription start site • Two 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 NELF 11-27 TFIIS Stimulates Proofreading of Transcripts • TFIIS stimulates proofreading, the correction of misincorporated nucleotides, likely by stimulating RNase activity of the RNA polymerase • This would allow polymerase to cleave off a misincorporated nucleotide and replace it with a correct one 11-28 11.2 Class I Factors • The preinitiation complex that forms at rRNA promoters is much simpler than the preinitiation complex for class II RNA polymerase • It involves polymerase 1 plus two additional transcription factors: – A core-binding factor, SL1 or TIF-IB – A UPE-binding factor, upstream-binding factor (UBF) or upstream activating factor (UAF) 11-29 The Core-Binding Factor • The core-binding factor, SL1, was originally isolated on the basis of its ability to direct polymerase initiation • SL1 also shows species specificity • This factor is the fundamental transcription factor required to recruit RNA polymerase I 11-30 Upstream-Binding Factor (UBF) • This transcription factor is an assembly factor that helps the core binding factor to bind to the core promoter element • It works by bending the DNA dramatically • Degree of reliance on UBF varies considerably from one organism to another • Human 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 UPE 11-31 Structure and Function of SL1 • Human SL1 is composed of TBP and three TAFs (TAFI110, TAFI63, TAFI48) which bind TBP tightly • These TAFs are completely different from those found in TFIID • Yeast and other organisms have TAFIs that are different from the human group 11-32 11.3 Class III Factors • In 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 studied • Transcription of all classical class III genes requires TFIIIB and TFIIIC • Transcription of 5S rRNA genes requires all three 11-33 TFIIIA • TFIIIA was the first eukaryotic transcription factor to be discovered • First member of the family of DNA-binding proteins that feature a zinc finger to be described – Zinc finger is roughly finger-shaped protein domain that contains 4 amino acids that bind a zinc ion – Has nine zinc fingers that appear to insert into the major groove on either side of the promoter for the 5S rRNA gene 11-34 TFIIIB and TFIIIC • Both of these transcription factors are required for transcription of the classical polymerase III genes • They depend on each other for their activities • TFIIIC is an assembly factor that allows TFIIIB to bind to the region just upstream of the transcription start site • TFIIIB can remain bound and sponsor initiation of repeated transcription rounds 11-35 Scheme for Assembly of the Preinitiation Complex on a classical class II promoter • TFIIIC binds to internal promoter • TFIIIC promotes binding of TFIIIB with its TBP • TFIIIB promotes polymerase III binding at start site • Transcription begins 11-36 Model of Preinitiation Complex on TATA-Less Promoter • Assembly factor binds first • Another factor, containing TBP, is now attracted • Complex now sufficient to recruit polymerase except for class II • Transcription begins 11-37 The Role of TBP • Assembly of the preinitiation complex on each kind of eukaryotic promoter begins with binding of an assembly factor to the 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 11-38