Eukaryotic Transcriptional Regulation (2024-25) PDF

Transcriptional regulation in eukaryotes 1 – How transcription is initiated Ben Nicholas Biol2010 2024-25 Semester 1 LO’s: By the end of this lecture you should be able to…. Describe how the pre-initiation complex (PIC) is assembled Outline the key ste...

Transcriptional regulation in eukaryotes 1 – How transcription is initiated Ben Nicholas Biol2010 2024-25 Semester 1 LO’s: By the end of this lecture you should be able to…. Describe how the pre-initiation complex (PIC) is assembled Outline the key steps in assembly of the PIC (initiation) Detail the post-initiation events that occur during transcription (elongation) Provide details of how core promoters and upstream regulatory elements (proximal and distal) help to regulate transcription Transcription Process of transfer of genetic (DNA) information into proteins RNA is a bit similar to DNA but is single stranded This means it can have functional tertiary structure How prokaryotes and eukaryotes differ How prokaryotes and eukaryotes differ  Eukaryotic transcriptional regulation overview Biochemistry, Miesfield and McEvoy, 2 nd Ed  Eukaryotic transcriptional apparatus Three types of RNA polymerase in eukaryotes Type of Polymerase Genes Transcribed RNA polymerase I 28s rRNA, 18s rRNA, 5.8S rRNA All genes that encode RNA polymerase II proteins, snoRNA, most snRNA 5S rRNA, tRNA, some snRNAs RNA polymerase III and genes for other small RNAs  Characteristics of eukaryotic transcription  RNA pol II requires general transcription factors (equivalent to s factor) to allow transcription to begin  Eukaryotic DNA is packaged into nucleosomes. As a result, transcription initiation is more complex. Complexity of the Eukaryotic system  Typical arrangement of a eukaryotic promoter region Upstream sequence elements Biochemistry, Miesfield and McEvoy, 2 nd Ed  Core promoters TATA containing Core promoter TATA less core promoter TATA INR INR DPE DPE usually +28 to +32 INR = initiator, a core promoter element (3’ to start site) DPE = downstream promoter element Motif AGAC Recognised by TF2I  Typical arrangement of a TATA core promoter Enhancer USE TATA Initiator (INR) BOX YYCAYYYY -1000 or more -100 -30 +1  Structure of eukaryotic TATA core promoters (recognized by RNA Pol II) Enhancer USE TATA Initiator (INR) BOX YYCAYYYY -1000 or more -100 -30 +1 Enhancer Upstream sequence Core Promoter elements  Core promoter: The initiator element (INR) Transcription start site TATA Initiator (INR) BOX YYCAYYYY  Simplest functional promoter  Can initiate basal transcription in absence of TATA box  Conserved Y is pyrimidine  Consensus sequence YYANWYY in humans, where, Y = C/T, W = A/ T, N=A/C/G/T, and +1 is underlined.  The INR element facilitates binding of TFIID.  Core promoter: The TATA box TATA Initiator (INR) BOX YYCAYYYY  Conserved sequence TATAAAA  Approx 25 bp upstream of initiator  Found in ~30% of promoters  Core promoter similar concept in Pribnow box (prokaryotes) and TATA box (eukaryotes). Complexity of the Eukaryotic system  All genes that are transcribed and expressed via mRNA are transcribed by RNA polymerase II. Similar in structure to bacterial RNApol Larger protein (12 subunits) Requires many transcription factors Initiation at the core promoter Need to assemble an RNApol II pre-initiation complex This positions the RNA pol II over transcription start sites. TFIID Coding strand 5’ 3’ TATA +1 Template strand Initiation General transcription factors for RNA Pol II (TFII) bind to the TATA box. The first transcription factor TFIID binds the TATA box Coding strand TFIID 5’ 3’ TATA +1 Template strand  Initiation The TFIID complex consists of TBP and TAF TAFs (TBP associated factors ~12). TAFs Involved in binding other TFs to help attach RNAP II TBP (TATA binding protein) to the DNA and attaching proteins that unwind the Coding strand chromatin structure. TFIID 5’ TB P 3’ TATA +1 Template strand Initiation TFIIB TFIIA Coding strand TFIID 5’ TB P 3’ TATA +1 Template strand Initiation TFIIB then recruits RNApol II to the TATA box. Coding strand TFIIB TFIIA TFIID 5’ TB P 3’ TATA +1 Template strand Initiation TFIIF accompanies the RNAPol II and stabilises it TFIIE and TFIIH bind and make up the initiation complex TFIIF RNA Pol II TFIIE Coding strand ATPase TFIIB TFIIA TFIID TFIIH Helicase 5’ TBP 3’ TATA +1 Template strand Initiation Pre- initiation complex (PIC) is now assembled  Initiation of Transcription TFIIF RNA Pol II TFIIE Coding strand TFIIB TFIIA TFIID TFIIH 5’ TB P 3’ TATA +1 Template strand Initiation Pre- initiation complex (PIC) is assembled  TFII H helicase activity melts the DNA TFIIF RNA Pol II TFIIE Coding strand ATPase TFIIB TFIIA TFIID 5’ TFIIH Helicase TBP 3’ TATA +1 Template strand Initiation Pre- initiation complex (PIC) is assembled  Transcription starts TFIIF RNA Pol II TFIIE Coding strand TFIIB TFIIA TFIID TFIIH 5’ TB P 3’ RNA Template strand  Elongation TFIIH  A 10 subunit complex containing: ATPase, Helicase, Protein kinase  Phosphorylation of the C terminal domain of RNApol II by TFIIH kinase activity occurs TFIIF RNA Pol II TFIIE C-terminal domain P TFIIB TFIIA P TFIID P TFIIH TB P RNA Lin et al 2011, Nature structural & molecular biology  Elongation After phosphorylation, TFs are released from the complex RNA Pol II C-terminal domain P P TFIID P TB P RNA Elongation  TFIIH is central to the elongation process  Conformation change – RNApol II tightens grip  Acquires new proteins – including elongation factors that help process the RNA and increase elongation rate RNA Pol II P TFIID P P TB P RNA So how is the assembly of RNApolII regulated to control transcription?  Structure of eukaryotic promoters (recognized by RNA Pol II) Mediator Pre- initiation complex (PIC) directs only a very low level of transcription Enhancer USE TATA BOX -1000 -100 -30 +1 Enhancer Upstream sequence Core Promoter elements  So how is Cell fate determined? Cell fate is determined by: Gene expression Unique sTF environment for Cell-cell/cell-matrix interactions each cell type External factors e.g. hormones  Structure of eukaryotic promoters (recognized by RNA Pol II) Pre- initiation complex (PIC) directs only a very low level of transcription Enhancer USE TATA BOX -1000 -100 -30 +1 Enhancer Upstream sequence Core Promoter elements  Upstream sequence elements Transcription can be enhanced by the binding of basal/upstream/specific transcription factors to sites ~100bp upstream of the PIC USE TATA Upstream sequence elements (USE) e.g., GC box & CAAT box – tissue specific enhancement of transcription.  USE promoter examples USE promoter Consensus sequence TF binding GC box GGGCGG Binds SP1 TF CAAT box GG(T/C)CAATCT. Binds CAAT box TF (CTF) e.g. C/EBP These must be in same orientation as the RNA pol II initiation site Upstream position is important. SP1 = specificity protein 1 C/EBP = CCAAT enhancer binding protein  Upstream sequence motifs 1. DNA sequence motifs bound by ubiquitous transcription factors e.g., the TF, Sp1 binds to GGGCGG Sp1 is found in all cell types 2. DNA sequence motifs that confer tissue specific expression e.g., MyoD binds to CANNTG (N=any base) MyoD is a muscle-specific transcription factor Note all cells have CANNTG but only tissue specific cells have the MyoD TF expressed. 3. DNA sequence motifs that confer response to particular stimuli e.g., Oestrogen receptor binds to AGGTCANNNTGACCT Structure of eukaryotic promoters (recognized by RNA Pol II) Enhancer USE TATA BOX -1000 or more -100 -30 +1 Enhancer Upstream sequence Core Promoter elements Enhancers  Regulatory sequences that act at a distance  Cis acting (up to 1Mb away) with reversible orientation  Bound by activator proteins  These interact with the mediator complex  Encourage binding of RNApol II Enhancer USE TATA BOX -1000 -100 -30 +1 Evidence for the existence of enhancers Simian Virus 40 (SV40) – promoter - found that the deletion of a 72 bp sequence led to a 100-fold decrease in expression Enhancer USE TATA BOX -1000 -100 -30 +1 Structure of eukaryotic promoters (recognized by RNA Pol II) Enhancer USE TATA BOX +1 Upstream Sequence Core Promoter Enhancer Elements  Properties of enhancers elements a) They can activate transcription when placed thousands of bp away from the TATA box Properties of enhancers elements Upstream Sequence Elements cannot be so far away. If Sp1 site placed more than 100bp away – does not work b) They act in either orientation 5’ to 3’ 3’ to 5’ Again this would not work for the Sp1 site c) Can act when placed upstream or downstream of the TATA box, or when placed within an intron Located within introns Indicator of the Importance of Introns That is one reason that intron polymorphisms may have effects although they are not translated.  Next time…..  How do we find transcription factors?  Exactly how do they work?  How are they regulated?  Can they be useful therapeutically?