Transcriptional Regulation in Eukaryotes 2 - Transcription Factor Structure and Regulation of Transcription PDF

Transcriptional regulation in eukaryotes 2 – Transcription factor structure and regulation of transcription Ben Nicholas Biol2010 2023-24 Semester 1 464616 Los: By the end of this lecture you should be able to… Describe how transcription factors are identifi...

Transcriptional regulation in eukaryotes 2 – Transcription factor structure and regulation of transcription Ben Nicholas Biol2010 2023-24 Semester 1 464616 Los: By the end of this lecture you should be able to… Describe how transcription factors are identified and purified Detail structural features that enable TFs to do their job List the three main types of DNA binding domains in TFs Give an example of a TF Describe the main ways that co- activators/repressors control transcription 464616  Transcription factors  Determine whether transcription occurs  Determine cell specificity  Confer response to specific timed stimuli  Structure is central, determined by the exact sequence of amino acids.  Mechanisms – how they initiate transcription can be difficult to determine due to TFs being expressed at low levels in cells. 464616  How do we find them?  Bioinformatics analysis of whole genome sequencing data can identify regions up/downsteam of genes containing conserved sequences.  Before that, mutation analysis (observational or interventional) of upstream regions to see what controls transcription  Eventually identify multiple conserved sequences which appear to regulate transcription in different 464616 genes  Isolation of transcription factors In 1986 Tijan isolated Sp1 by DNA affinity chromatography Cervical cancer cells taken in 1951 from Henrietta Lacks https://www.bbc.co.uk/ideas/videos/how-one-womans-immortal-cells-changed-the-world/ doi: 10.1073/pnas.83.16.5889 p08wr9gf 464616  Isolation of transcription factors In 1986 Tijan isolated Sp1 by DNA affinity chromatography Adapt DNA sequence for any transcription factor Cervical cancer cells taken in 1951 from  More TFs identified Henrietta Lacks 464616  Transcription factor structure  TFs are proteins made up of amino acids hence their 3D structure is important to their function.  Modular structure one region binds DNA another region binds to other components e.g., Oct-2 an octamer transcription factor, specific for B-cells The members of this subfamily (designated Oct-l, Oct-2, Oct- 3, etc.) recognize an evolutionarily conserved octanucleotide sequence in the vertebrate promoter and enhancer elements (5′-ATGCAAAT-3′). 464616  Transcription factor structure  TFs are proteins made up of amino acids hence their 3D structure is important to their function.  Modular structure - All the amino acids for DNA binding in 1 region e.g,. Oct-2 60aa 467 aa Full length Oct2 60aa Same affinity 464616  Transcription factors TFs have different domains for different functions  DNA binding domains  Activation domains OR Inhibitory domains 464616  DNA binding domains 1. Zinc fingers 2. Helix turn helix 3. Basic binding domains Binding mostly based on a-helices fitting into DNA grooves 464616 1. Zinc finger domains 2+  Contains a loop of 23 aa  Usually have multiple zinc fingers per TF  The linker between the fingers is 7-8 aa  a-helix contacts the major groove of DNA 464616 Often multiple zinc fingers involved in binding the specific DNA sequence. Zn2+ ion does not directly interact with the DNA but is essential for the folding of the finger. Zinc fingers bind both to the major and minor grooves. 464616  SP1 is a zinc finger TF From: Human pdb SP1, Uniprot 2. Helix turn helix Two helices held at a fixed angle Recognition helix binds major groove of DNA  Bind DNA as dimers, so the 2 recognition helices are separated by one turn of the DNA helix 464616  Homeodomains contain H-T-H based structures Homeodomain 2 COOH 3 1 NH2  Homeodomains are 60 aa and contain 3 -helices The C terminal -helix #3 is 17 a/a’s and lies in the major groove  Helices 1 & 2 point away from the DNA  H-T-H can be di-helical, trihelical, tetrahelical etc TFIIE is a tetramer of this tri-helical H-T-H protein. 3. Basic (+ve charged) binding domains  Transcription factors with basic binding domains cannot bind to DNA alone  Transcription factors with basic binding domains must dimerise Must have dimerisation motif + + + - + + -- - + + + + -- + NH2 - NH2 - -- - -- - Structure:function of basic binding domains Leucine zipper Helix-loop-helix  C/EBP is a basic zipper TF From: Human pdb C/EBP, Uniprot  Summary of DNA binding domains DNA binding Transcription domain factor Most TFs have one of these three types of binding domain: 1. Zinc fingers 2. Helix turn helix 3. Basic binding domains  Transcriptional factor activity may be regulated by location if responsiveness is important e.g. Steroid hormone receptors - Cys2-Cys2 zinc fingers - Steroid hormones are synthesised in response to a variety of neuroendocrine activities - They exert major effects on cell growth, tissue development and body homeostasis e.g. Vitamin D receptor (VDR) Glucocorticoid receptor (GR) DNA binding domains have 43–97 % Estrogen receptor (ER) sequence homology Androgen receptor (AR Vitamin D Cytoplasm Nucleus VDR VDR DNA Inactive form TGTTCT TGTTCT VD responsive elements Vitamin D Cytoplasm Nucleus VDR VDR DNA TGTTCT TGTTCT VD responsive elements Vitamin D Cytoplasm Nucleus VDR VDR DNA TGTTCT TGTTCT Conformation change VD responsive elements Form dimer Move to nucleus Vitamin D Glucocorticoids activate GR Estrogens activate ER Androgens activate AR Cytoplasm Nucleus All inactive in TXN VDR VDR cell until ligand DNA arrives TGTTCT TGTTCT VD responsive elements Vitamin D receptor structure How do transcription factors activate transcription? e.g. Oct-2 60aa 467 aa Full length Oct2 60aa No effect on transcription Activates transcription  Activation domains DNA binding Activation or domain Inhibitory domain A region of the transcription factor protein involved in activating/inhibiting transcription  Domain swap experiment Transcription Factor 1 Transcription Factor 2 (well characterised) (unknown activation domain) Gal4 DNA Binding A B C domain Combine DNA-binding domain of factor 1 with different regions of factor 2 A B C Test on a gene carrying the binding site for factor 1  Domain swap experiment Test on a gene carrying the binding site for factor 1 A B C - + - Therefore domain B has activation domain of Factor 2 Gal 4 TATA Binding site Different types of activation domains Activation domain a) Acidic amino acids e.g., Gal 4 b) Glutamine rich No specific structure e.g., Oct-2 – just sequence c) Proline rich e.g., Jun  How do TF activation domains work? Pre- initiation complex (PIC) is assembled TAFs (TBP associated factors) interact with the activation/inhibitory domains TFIIF RNA Pol II TFIIE TFIIB TFIIA TFIID TFIIH TB P TATA +1 TBP (TATA binding protein) Structure of eukaryotic promoters (recognized by RNA Pol II) Enhancer Upstream Sequence Core Promoter Elements Both the USE TF and the enhancer TF have their activating domains interacting with the TFIID  Two key ways transcription activation factors can work: a)Through direct interaction with the PIC Transcription factor TAF domain Sp1 TAF110 Oestrogen Receptor TAF30 b) Through the recruitment of co- activators i) Co-activators work by interacting with the PIC ii) Or by opening/loosening chromatin structure TF PIC  Co-activators can alter chromatin structure TFs recruit co-activators to modify histones  Histones: H2a, H2b, H3 and H4 Transcription factor  -vely charged DNA wrapped around the +vely charged histones  Histones have 2 domains  - globular domain  - amino tail domain very rich in lysines (+charge)  Co-activators can alter chromatin structure TFs recruit co-activators to modify histones  Histone acetyltransferase (HAT) Co-activator - Acetylates N-terminal tail lysine of histone units TF - Neutralizes +ve charge of histone  Opens up DNA  Allows transcription factors & RNA polymerase II to get to the DNA Activating transcription factors recruit co-activators which modify the histones  e.g., Glucocorticoid receptor (GR)  Recruit the co-activator p300/CBP  p300/CBP has histone acetyl transferase (HAT) activity - p300/CBP will acetylate: H3 H4 H2A H2B TRANSCRIPTION  Inhibitory domains Also need a way to switch genes off Like bacterial repressors DNA binding Inhibitory domain domain A region of the transcription factor protein involved in repressing transcription How do TF inhibitory domains work? a) Bind to DNA and block TFs with activator domains, from binding b) Bind to PIC and block transcription with its inhibitory domain (a) and (b) act by getting in the way c) Through the recruitment of co-repressors i) Co-repressors work by interacting with the PIC ii) Closing / tightening chromatin structure  Co-repressors can alter chromatin structure By modifying histones  Histone de-acetylase (HDAC) - Removes acetyl group of histone units - restores +ve charge of histone  Close down DNA  Shutting off transcription  SMRT is a co-repressor  Forms a large co-repressor complex with RAR or TR and HDAC 1&2 in the absence of ligand, stabilising their interaction with TFIIB  Mediates the repressive activity of unliganded nuclear receptors  Tamoxifen for breast cancer Oestrogen receptor antagonist Induces ERa conformational change which favours co-repressor (SMRT or N-CoR) binding Some breast cancers show co-activator recruitment even in the presence of tamoxifen, making them resistant to the treatment  Summary  Transcription factors bind directly to DNA and have another domain that influences transcription  Three main types of DNA binding domain  The activation/repressor domain is separate and either binds directly to RNApolII or works through co-activator or co-repressor  TF inhibitory domains work by preventing TF binding, or binding to PIC with inhibitor domain and preventing transcription directly repressing it, or by recruiting co-repressors, modifying histone structure by tightening it  TF activation domains work by interacting with the PIC or by recruiting co-activators, modifying histone structure by loosening it  Transcriptional regulation in eukaryotes 3 – Post- transcriptional modification Ben Nicholas Biol2010 2023-24 Semester 1 LO’s: At the end of this lecture you should be able to: Summarize the key events in post- transcriptional processing of RNA Outline the function and structure of 5’ capping Detail the mechanism by which polyadenylation of the RNA transcript occurs Describe how 5’ capping and polyadenylation are subject to quality control mechanisms Difference between prokaryotes and eukaryotes In prokaryotes, transcription and translation occur in the same compartment In eukaryotes, transcription is purely in the nucleus and translation in the cytoplasm This allows greater degree of control over these processes Presents some challenges too  Elongation  C-Terminal domain (CTD) of RNA Pol II phosphorylated  CTD – 52 tandem repeats of 7 amino acids - Each contains 2 serines (Ser2 & Ser5) - Phosphorylated  Phosphorylation allows loading of RNA processing machinery RNA Pol II e.g., 5’ modification enzymes, elongation factors, splicing proteins, P 3’ modification enzymes, TFIID P protection P TB P RNA  Termination  Unlike in prokaryotic transcription, eukaryotic transcription via RNApol II extends up to 2K nucleotides past the end of the gene  The other polymerases have more clearly defined endpoints.  Additional nucleotides are clipped from the end of the pre-mRNA after transcription has ceased as part of mRNA processing  Post-transcriptional processing The RNA transcript has to be tagged and modified to identify it as mRNA that will ultimately be used to produce protein mRNA processing is tightly coupled to transcription Post-transcriptional control adds layers of complexity to gene expression. Important in quality control of mRNA (only intact messages Nuclear events should be exported). Cap and tail help maintain stability. Recognition of these and e.g., EJC proteins, allows export from nucleus.  Post-transcriptional processing 5 Major Alterations to the primary RNA transcript: 1. Addition of 5’ Cap – protection and marking a mRNA. 2. Splicing - to remove non-coding regions 3. 3’ processing and polyadenylation – marking as the end of the mRNA and protection. 4. Editing (rare but important) – similar to mutating the mRNA to produce alternative final product but this is not a random event. 5. Transport – to cytoplasm for translation.  1. Addition of 5’ cap Simple eukaryotes Multicellular eukaryotes The 5’ cap is guanosine with a methyl group on the 7-position (m7G) Essential for efficient translation, stabilisation and transport of mRNA From: Takara bioscience  Addition of 5’ cap The 5’ cap is guanosine with a methyl group on the 7-position (m7G) Essential for efficient translation, stabilisation and transport of mRNA  Addition of a 5’ Cap  The first thing that g b a happens to the RNA is addition of a 5’ cap  Addition of 7 methyl- guanosine to the 5’ end of RNA  The RNA is capped as Unusual 5’-5’ soon as it emerges from linkage resulting in the exit channel of RNA a molecule with polymerase II (~25-30 effectively two 3’ bp) ends. Cells contain many nucleases that cleave at the 5’ ends.  Addition of a 5’ Cap  Capping enzymes bound to the RNAP II’s C- g b a terminal domain (CTD).  The first base at 5’ end of the RNA contains three phosphates: alpha, beta, gamma  1. RNA triphosphatase removes the gamma- phosphate  Addition of a 5’ Cap  Capping enzymes bound to the RNAP II’s C- g b a terminal domain (CTD).  The first base at 5’ end of the RNA contains three phosphates: alpha, beta, gamma  1. RNA triphosphatase removes the gamma- phosphate  Addition of a 5’ Cap  GMP is added to the 5’ end of the RNA a b g GTP  2a. Guanylyltransferase - Removes the gamma and beta phosphates of GTP - GMP (guanosine monophosphate)  Addition of a 5’ Cap  GMP is added to the 5’ end of the RNA a GTP  2a. Guanylyltransferase - Removes the gamma and beta phosphates of GTP - GMP (guanosine monophosphate)  Addition of a 5’ Cap  2b. Guanylyltransferase - Then the GMP (guanine) is added to the 5’ terminal base of the transcript  G is added to the RNA in reverse orientation from all other nucleotides forming a 5’-5’ linkage  Addition of a 5’ Cap  Guanylyltransferase - The GMP (guanine) is added to the 5’ terminal base of the transcript  3. Methyltransferase - Adds methyl group to Other methyl groups may guanine (7th pos of the purine) be added but m7G cap is the most abundant.  The CTD is central to this process The highly phosphorylated CTD of RNAP II provides the binding sites for proteins involved in the post-transcriptional modification of the growing RNA transcript. Co-transcriptional capping 25 1 2 3 1. RNA triphosphatase 2. Guanylyltransferase 3. Methyltransferase  Addition of a 5’ Cap – Why? Addition of 7 methyl-guanosine to the 5’ end of RNA Step 1 Step 2 Guanylyltransferase methyltransferase 5’ 5’ & phosphatase 5’ --- 5’ Gppp + pppRNA GpppRNA 7Me GpppRNA (GTP) (GMP)  The cap helps to distinguish mRNA from other RNA  Helps mRNA be properly processed and exported from the nucleus  Protects it from degradation in the cell Addition of a 5’ Cap – Why? The 5' cap of eukaryotic messenger RNA is bound at all times by various cap-binding complexes (CBCs). CBC aids in the export of the mRNA and protect it from decapping. They also serve as a marker for the pioneer round of translation when the message is examined by nonsense mediated decay (mRNA quality assurance)  Quality control of 5’ capping  Improperly capped RNA is recognised by quality control mechanisms and becomes degraded  Mechanism in yeast is Rai1-Rat1 heterocomplex. Rai1 decaps improperly capped RNA. Rat1 has exoribonuclease activity and degrades the uncapped RNA.  Mammalian DXO in the nucleus performs decapping and also degrades uncapped RNA and RNA with an unmethylated cap  How do viruses get their mRNA made?  Occurs in negative, ssRNA viruses  Influenza virus cap snatching occurs in the cell nucleus  First 10-20 nucleotide residues are stolen from host mRNA  Viral RNA-dependent RNA polymerase (RdRp) the transcribes positive sense viral mRNA including 5’ terminal extension that is not coded in the viral genome  The de-capped host mRNA is targeted for degradation leading to downregulation of cellular mRNA.  Steps in influenza cap snatching Viral RdRp has three subunit: PA, PB1 and PB2 Antiviral drug baloxavir marboxil inhibits the endonuclease function of the PA subunit, preventing transcription 2. RNA splicing Prokaryotes Eukaryotes Polycistronic: Monocistronic: mRNA encodes mRNA encodes a two or more single protein, proteins, no intronic introns  Introns  First detected in 1977  Function unknown but likely to allow generation of multiple products from the same gene. Average human gene contains 8-9 introns Evolutionarily conserved genes contain more introns* Gene Length (kb) Number of introns Insulin 1.4 2 b-Globin 1.4 2 Serum albumin 18 13 Type VII collagen 31 117 Factor VIII 186 25 Dystrophin 2400 78 #Adapted from Strachan and Read (1996) *Gorlova et al, 2014doi: 10.1186/1471-2148-14-50  3 classes of RNA splicing 1. Nuclear pre-mRNA splicing  Most eukaryotic genes (common)  Two transesterification reactions (branch site A)  Spliceosomes 2. Group II self-splicing  Some eukaryotic genes (rare)  e.g. rRNA, organellar mRNA or fungi, plants and bacteria  Two transesterification reactions (branch site A)  RNA enzyme encoded by intron (ribozyme) 3. Group I self-splicing  Some eukaryotic genes (rare)  e.g. introns in eukaryotic viruses  Two transesterification reactions (branch site G)  RNA enzyme encoded by intron (ribozyme)  How are introns removed? Conserved sequence motifs indicate exon/intron boundaries RNA Intron 5’ Exon Exon 3’ GU AG branch 5’ splice 3’ splice point site Site Start of intron End of intron  Mechanism of RNA splicing 5’ Splice Site Start of intron Further conserved AG GUAAGU sequence motifs at the Exon Intron GU and AG sites 3’ Splice Site End of intron (Y6)(N6)AG...... Y =pyrimidines (T or C) Branch point Key to splicing UACUAAC reaction  Mechanism of RNA splicing Introns are removed by 2 transesterification reactions Step 1 : cleavage at the 5’ splice site  The backbone hydroxyl group (OH) of the A within the branch point acts as a nucleophile and attacks the 5’ splice site  Mechanism of RNA splicing Introns are removed by 2 transesterification reactions Step 1 : cleavage at the 5’ splice site  The ribose hydroxyl group (OH) of the A within the branch point acts as a nucleophile and attacks the 5’ splice site 5’ SS 3’ SS  Mechanism of RNA splicing  Intron folds back on itself  Phosphodiester bond between A (BP) and G (5’SS) 5’ SS 3’ SS 5’ SS 3’ SS -OH  Mechanism of RNA splicing Step 2: cleavage at the 3’ splice site and joining of the exons  The newly liberated 3’OH group of the 5’ exon becomes a nucleophile and attacks the phosphoryl group at the 3’ splice site 5’ SS 3’ SS  Mechanism of RNA splicing 2 steps and both go through Cleavage at 3’ splice site ‘cut and join’ 5’ SS 3’ SS Exons ligated together Lariat Intron released & degraded in cell  Summary Addition of a 5’ cap helps to stabilise the mRNA and earmark it for transport out of the nucleus 5’ cap protects mRNA from degradation in the cell Eukaryotic genes contain introns which must be excised from the pre-mRNA This removal of introns occurs by RNA splicing RNA splicing occurs at conserved motifs in the RNA sequence by a two-step process