Molecular Biology Fifth Edition 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)

11.1 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

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)

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

Model of Formation of the DABPolF Complex

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

Structure of the TBP-TATA box complex

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

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

Model for the Interaction Between TBP and Promoters

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

Transcription Enhancement by Activators

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 (TBP-related factor 1) The general transcription factor NC2: Stimulates transcription from DPE-containing promoters Represses transcription from TATA-containing promoters

Structure and Function of TFIIB Structural studies have revealed that TFIIB binds to TBP at the TATA box via its C-terminal 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

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

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”

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 carboxyl-terminal 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

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

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

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

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

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

Model for the participation of GTFs in initiation, promoter clearance, and elongation

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

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

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

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

11.2 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)

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

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

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

11.3 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

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

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

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

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

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