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Molecular Biology Lecture 17 Chapter 11 General Transcription Factors in Eukaryotes Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 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 11-2 The Class II Preinitiation Complex • Class II preinitiation complex contains: – Polymerase II – 6 general transcription factors: • • • • • TFIIA TFIIB TFIID TFIIE TFIIH • The transcription factors (TF) and polymerase bind the preinitiation complex in a specific order 11-3 Model of Formation of the DABPolF Complex 11-4 11-5 The polymerase extends the footprint to +17 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 – 8 to 10 copies of TBP-associated factors (TAFIIs) specific for class II 11-7 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 11-8 TBP-TATA box complex Overall bend of 80 degrees on the DNA caused by TBP11-9 TBP In-vitro transcription assays 11-10 The TBP-Associated Factors • These are also called TAFIIs • 8 different proteins are designated by MW • Most are evolutionarily conserved in eukaryotes • 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 11-11 Structure of TFIID 11-12 Most TAF proteins Are conserved from Yeast to human 11-13 Adenovirus promoters In-vitro transcription assays Requirement of TAFs for efficient transcription on AdML 11-14 and Hsp70 promoters Roles of TAFII250 and TAFII150 • The TAFII250 and TAFII150 help TFIID bind to the initiator and DPE of promoters • Also aid in TFIID interaction with Sp1 that is bound to GC boxes upstream of the transcription start site • They enable TBP to bind to: – TATA-less promoters that contain elements such as a GC box • TAFII250 has 2 enzymatic activities: – Histone acetyltransferase – Protein kinase 11-15 DNAse 1 footprinting on the hsp70 promoter 11-16 bhTBP = TBP produced in bacteria vhTBP = TBP produced in virus infected cells 11-17 Activation by SP1 requires TAF 110 11-18 Model for the Interaction Between TBP and Promoters 11-19 Transcription Enhancement by Activators 11-20 Structure and Function of TFIIB • The 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 domain – 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 11-21 Function of TFIIB – Binds to TBP and recruits polymerase Function of TFIIA – Stabilizes TBP-TATA box binding 11-22 Function of TFIIF – Made of two protein RAP30 and 70 RAP 30 sigma-like binds to E. coli polymerase required for Binding of RAP70 to core enzyme. TFIIF reduces non-specific interaction of polymerase with DNA 11-23 TFIIH • TFIIH is the last general transcription factor to join the preinitiation complex with TTIIE • Plays 2 major roles in transcription initiation: – Phosphorylate the CTD of RNA polymerase II – Unwind DNA at the transcription start site to create the transcription bubble 11-24 Phosphorylation of the CTD of RNA Polymerase II • The preinitiation complex forms with hypophosphorylated form of RNA polymerase II • 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 11-25 of transcription Function of TFIIH (9 subunits) And TFIIE 11-26 Function of TFIIH – Phosphorylation of CTD of PolII 11-27 The TFIIH helicase activity is required for transcription in yeast Permissive temperature Nonpermissive temperature 11-28 The Elongation Factor TFIIS • Eukaryotes control transcription primarily at the initiation step • There is some control exerted at elongation • TFIIS, isolated from tumor cells, specifically stimulates transcription 11-29 TFIIS stimulates elongation 11-30 TFIIS Stimulates Proofreading of Transcripts • TFIIS stimulates proofreading, 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 • Proofreading is the correction of misincorporated nucleotides 11-31 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 11-32 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 11-33 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 11-34 Schematic Model 11-35 Class I Factors • RNA polymerase I and III transcription factors make up the preinitiation complex, much simpler than the preinitiation complex for class II RNA polymerase • Transcription factors: – A core-binding factor, SL1 or TIF-IB – A UPE-binding factor, upstream-binding factor (UBF) or upstream activating factor (UAF) 11-36 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 11-37 Upstream-Binding Factor • This transcription factor is an assembly factor that helps SL1 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 • Size of polypeptide is 97-kD 11-38 Structure and Function of SL1 • Human SL1 is composed of TBP and TAFs which bind TBP tightly: – TAFI110 – TAFI63 – TAFI48 • These TAFs are completely different from those found in TFIID • Yeast and other organisms have TAFIs that are different from the human group 11-39 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 11-40 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 11-41 Scheme for Assembly of Preinitiation Complex • TFIIIC binds to internal promoter • TFIIIC promotes binding of TFIIIB with its TFB • TFIIIB promotes polymerase III binding at start site • Transcription begins 11-42 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 11-43