UC Biologia Molecular Past Paper 2024-2025 PDF

UC Biologia Molecular (40395)| Ano lectivo 24_25 Parte II| Aula: 1 12 e 13 novembro SUMÁRIO Mecanismos moleculares do processo de transcrição em eucariotas. Polimerases de eucariotas (I, II e III) Interacções DNA-proteína e proteína-proteína; reconhecimento do promotor pelas RNA polimerases. TBP é...

UC Biologia Molecular (40395)| Ano lectivo 24_25 Parte II| Aula: 1 12 e 13 novembro SUMÁRIO Mecanismos moleculares do processo de transcrição em eucariotas. Polimerases de eucariotas (I, II e III) Interacções DNA-proteína e proteína-proteína; reconhecimento do promotor pelas RNA polimerases. TBP é um factor quase universal. Associação do aparelho de transcrição no promotor. Iniciação da transcrição. Processamento co-transcricional. Domínio CTD da polimerase II. Complexo cap e terminais 3´ do mRNA. O papel dos ativadores: elementos bi-direccionais que ajudam na iniciação. Papel dos enhancers. A transcrição é um processo coordenado envolvendo vários fatores proteicos. Molecular mechanisms of the transcription process in eukaryotes. Eukaryotic polymerases (I, II and III) DNA-protein and protein-protein interactions; Promoter recognition by RNA polymerases. TBP is an almost universal factor. Association of the transcription apparatus in the promoter. Start of transcription. Co-transcriptional processing. CTD domain of polymerase II. Cap complex and 3' ends of mRNA. The role of activators: bi-directional elements that help with initiation. Role of enhancers. Transcription: a coordinated process involving several proteic factors. 1 S. Mendo| BM24_25 Major di erences between prokaryotic and eukaryotic transcription ‣ Takes place on a densely proteinized chromatin template ‣ Bacterial RNA pol reads the DNA whereas eukaryotic RNA pol cannot read DNA 2 S. Mendo| BM24_25 ff Two models explain eukaryotic gene transcription Two Models: Enhanceosome (nucleoprotein complexes comprising multiple TFs) vs Hit and run (master TFs binds the promoter, initiates transcription and leaves) 3 S. Mendo| BM24_25  Eukaryotic transcriptional regulation ✓The genomes of multicellular eukaryotes folds into Topologically Associated Domains (TADs); ✓Chromosomes with low gene density reside at the nuclear periphery; chromosomes with high gene density, occupy the nuclear interior; ✓Transcriptionally active genes associate with nuclear pore complexes that span the nuclear pore. (why??) Transcription decondenses chromosome territories; the loops collapse back into condensed territories when transcription ceases. https://doi.org/10.1016/S0092-8674(03)00078-3 The banding pattern of polytene chromosomes corresponds to TADs 4 S. Mendo| BM24_25  Nuclear architecture and premature aging syndromes ✓The link between disrupted nuclear membrane and premature aging syndromes highlights the importance of the spatial organization of the nucleus. ✓Progeria Patients cells: Nuclear lamina: protein meshwork underlying the nuclear membrane ‣ altered nuclear size and shape (inside the nucleus of eukaryotic cells); composed of intermediate laments proteins lamin A, B and C. It provides: ‣ disrupted nuclear membranes i) mechanical support ‣ altered redox homeostasis (NRF2 a ii) regulates cellular events (DNA replication and cell division) redox sensor gets trapped at nuclear iii) participates in chromatin organization periphery, resulting in impaired NRF2 iv) anchors the nuclear pore complexes embedded in the nuclear signalling, chronic oxidative stress - envelope, being a platform for assembly of protein complexes >>ROS- and genomic instability) involved in signal transduction pathways. ‣ Sirtuin 6 (SIRT6), a protein involved in v) gene expression (sequestering TFs at the nuclear envelop) promoting longevity ends up trapped inactive at the nuclear membrane. 5 S. Mendo| BM24_25 fi  Eukaryotic gene expression| Transcription Transcription is mediated by the collective action of: ‣ sequence-speci c DNA-binding TFs ‣ core RNA pol II transcriptional machinery ‣ various coregulators that bridge the DNA-binding TFs to the transcriptional machinery ‣ various chromatin remodelling factors that mobilize nucleosomes ‣ variety of enzymes that catalyse covalent modi cations of histones and other proteins 6 S. Mendo| BM24_25 fi fi  Eukaryotic gene expression| Transcription Eukaryotic transcription is a multistep process involving: Opening the chromatin Binding of BTF´s Binding the polymerase Eukaryotic transcription is most often under positive regulation ✓Polymerase binds to promoter ✓TFs bind to enhancers Comparison of a simple unicellular (yeast) eukaryotic promoter (a), and a diversi ed metazoan regulatory module (b) 7 S. Mendo| BM24_25 Eukaryotic gene expression| Transcription ‣ All genes are inactive as a default state ‣ They require the presence of constitutive transcriptional activators for expression ‣ TF´s are required to transcribe the cromatin template ‣ TF´s are modular proteins (consisting of a number of domains required for initiation of transcription) which are not part of the RNA polymerase ‣ TF´s (trans-acting), recognise the promotor DNA sequences (cis-acting); ‣ Eukaryotic gene expression is usually controlled at the level of initiation of transcription by opening the chromatin. 8 S. Mendo| BM24_25  How transcription factors act? ‣ Transcription factors act in four ways: i) by binding to DNA and interacting with the basal initiation apparatus; ii) by binding to DNA and altering its structure or conformation; iii) indirectly, by binding to DNA and interacting with another regulator to in uence its activity; iv) through protein-protein interactions (i.e. without binding to DNA at all), either directly with components of the basal apparatus or indirectly with other regulators. Some eukaryotic TF function by introducing bends into DNA, which facilitates the interaction of other components. protein-protein interactions Enhanceosome (coactivators and corepressors) 9 S. Mendo| BM24_25  Nucleic acid recognition by proteins ‣ DNA (dsDNA)-binding proteins categories These incorporate domains that facilitate binding to nucleic acid Zinc- nger 2 anti-parallel Beta sheets and an alpha helix, separated by a short peptide that assumes a Helix-turn-helix HTH motif nger- like structure, which binds 2 alpha helices, separated by the major grove of a short Beta-turn; the DNA second interacts with the major grove of DNA ✓Interacting with DNA ends (e.g. DNA ligases, exonucleases) DNA-processing ✓Enclosing DNA or binding it in a deep crevice (e.g. DNA polymerases, enzymes topoisomerases) (possess a motif which ts into the major grove: increases stability of binding) ✓Interacting with the face of the helix. E.g.: ‣ most transcription factors, ‣ restriction enzymes ‣ DNA-packaging proteins ‣ site speci c recombinases ‣ DNA-repair enzymes. 10 S. Mendo| BM24_25 fi fi fi fi  Long range regulatory elements Regulatory DNA sequences that can work over distances of 100Kb or more from the gene promotor. enhancers (typical mammalian genome contains 106 enhancers, only 1-2% contribute to gene regulation; have between 100 and 500 bp; can be found anywhere including within introns; contain binding sites for different TFs; increase gene promotor activity in a tissue-speci c or developmental stage-speci c manner) silencers (between 700 and 1000 bp; can be found anywhere including within introns; decrease gene promotor activity) Transvection involves long-range interactions to bring enhancers in trans to their promoters. insulator (DNA element 300-2000bp that marks the border between regions of hetero and euchromatin; prevent inappropriate cross activation/repression of neighbouring genes - blocking enhancers ad silencers) locus control regions (LCRs) DNA sequences that enhance the transcription of downstream genes. 11 S. Mendo| BM24_25 fi fi  Initiation in eukaryotic promotors ‣ Various basal transcription factors (BTF´s) must bind to cis-acting elements to form the initiation complex at all promoters; only then RNA polymerase can bind to the core promotor (shortest sequence at which an RNA pol can initiate transcription) ‣ Basal factors are identi ed as TFNX, where N is I, II, or III (signifying the polymerase) and X is a letter (A, B.)… E.g.: Core promotor motifs for RNA polymerase II TBP: TATA binding protein TATA box: Major subunit of TFIID complex 12 S. Mendo| BM24_25 fi  Eukaryotic RNA polymerases and promotor recognition Eukaryotes have multiple nuclear DNA-dependent RNA Polymerases and organelle speci c RNA polymerases Cellular Product RNA polymerase compartment trancribed I Nucleolus 18S, 28S and 5.8S rRNA mRNA, some small nuclear (sn)RNA, small nucleolar (sno)RNAs (noncoding RNAs: U1- II Nucleus U7, except U6), repeated sequences (AluI elements), and micro (mi)RNAs tRNA, 5S rRNA, other snRNA (U6), 7SL III Nucleus RNA * Plants: nuclear RNA pol IV e V ‣ Promotors for RNA pol I and II: mostly upstream of the starting point (+1) ‣ Promotors for RNA pol III: downstream (within the transcription unit) characteristic short conserved sequences recognized by BTFs 13 S. Mendo| BM24_25 fi  RNA polymerases II promotors ‣ Enhancers and silencers can increase or inactivate, respectively, the expression of a gene ‣ Silencers bind negative regulators ✓ A cis-acting sequence ✓ increases the utilization of (most) eukaryotic promoters ✓ can function in either orientation and in any location (upstream or downstream) 14 S. Mendo| BM24_25  Enhancers assist initiation ‣ Enhancer activates an accessible promoter (with no intervening insulator) and can be virtually at any distance either upstream or downstream of the promoter ‣ Enhancer bind TFs (activators or repressors) ‣ Enhancer increases the concentration of activators in the vicinity of the promoter in cis, the more activators bound the higher the expression level Enhancer binding activators ✓ True activators: bind speci c DNA sites ✓ Those that recruit chromatin modi cation enzymes ✓ Architectural factors that bend the DNA ✓ Yeast UAS (analogous to enhancers): behaves like an enhancer but works only upstream of the promoter 15 S. Mendo| BM24_25 fi fi  How enhancers work ‣ Enhancers usually work only in cis con guration with a target promoter. ‣ Enhancers can be made to work in trans con guration by linking the DNA that contains the target promoter to the DNA that contains the enhancer via a protein bridge or by catenating the two molecules. ‣ The principle is that an enhancer works in any situation and at any distance from the promoter. ‣ Enhancers increase the concentration of activators in the vicinity of promoters in cis 16 S. Mendo| BM24_25 fi fi  Role of coactivators and corepressors ‣ Coactivators and corepressors are proteins that increase or decrease, respectively, transcriptional activity without binding DNA directly; they bind directly to TFs and i) recruit other proteins or ii) have enzymatic activity. ‣ Coactivators are divided into 2 classes: chromatin modi cation complexes (histone modi cations (writers- create modi cations): acetylation, methylation, ubiquitination, phosphorylation, ADP ribosylation, sumoylation) chromatin remodelling complexes (DNA unwinding activity: nucleosome sliding, remodelled nucleosome, nucleosome displacement, nucleosome replacement) In eukaryotes, all genes are inactive as a default state because they require the presence of constitutive transcriptional activators for expression. 17 S. Mendo| BM24_25 fi fi fi  Eukaryotic RNA polymerases ‣ Typically have 12 subunits (Rbp 1-12); 2 large and many smaller (some common to all polymerases) ‣ Aggregates of 500 KDa ‣ RNA pol II is the best characterised ‣ The largest subunit in RNA polymerase II has a carboxy-terminal domain (CTD): multiple repeats of a consensus sequence of 7 aa (26 in yeast; 52 in mammals) ‣ None of the RNA polymerases recognise their promotor directly ✓ regulates initiation reaction ✓ transcription elongation ✓ mRNA processing ✓ export of mRNA to the cytoplasm Synthesizes most of Synthesizes almost 1/4 of heterogeneous nuclear RNA 96% of all RNA; the cytoplasmic RNA (hnRNA)- precusor of most mRNA; Pol I is very active mRNA only 2-5% of total RNA 18 S. Mendo| BM24_25  Phosphorylation of the RNA polymerase II C-terminal domain (CTD) ✓CTD has a unique sequence of seven repeating residues (Y1S2P3T4S5P6S7), of which the number of repeats may differ depending on species (yeast: 26 repeats; mammals: 52). PAUSING (Unphosphorylated CTD) (Phosphorylated CTD) INCREASE of Ser2P 10.1039/D1CB00083G 19 S. Mendo| BM24_25  Mitochondria and chloroplast RNA polymerases ‣ Smaller; resemble bacterial polymerases ‣ Genomes are smaller, though less genes to transcribe and regulation is simpler. ‣ e.g.: plants Chloroplast RNA polymerase: i) plastid-encoded RNA polymerase (PEP; left panel): is a bacterial-type multi-subunit RNA polymerase composed of the core enzymatic subunits α, β, β , β (blue) and a sigma subunit (red) that is responsible for promoter recognitio ii) nucleus-encoded plastid RNA polymerase (NEP; right panel). 20 n  RNA polymerase I ‣ Directs RNA synthesis (in the nucleoli) Non-transcribed spacers (NTS) Precursor 28S + 18S transcript | Transcription unit | 21 S. Mendo| BM24_25  RNA polymerase I Accurate and promoter-speci c initiation of transcription requires: ✓ RNA Pol I ✓ upstream binding factor (UBF) ✓ selectivity factor 1 (SL1) (unusual for (Also G-C rich) a promotor) Promotor: 2 regions (core promotor, and (and to core promotor in humans) UPE which a ects e ciency of transcription Protein-protein interaction SL1 ‣ Also known as TIF-IB ‣ Is a multimeric protein composed of TBP (TATA-Binding Protein) and three TAFI (TBP-Associated Factors)- SL1 resembles bacterial sigma factor ‣ Recruitment of RNA Pol I ‣ Ensure that Pol I is placed at the start point ‣ Enable low level transcription even if UBS is absent RNA Pol I binds to TIF-IA, which bridges the polymerase and SL1 at the core promotor 22 S. Mendo| BM24_25 ff ffi fi  RNA polymerase III promotor organization ‣ Uses 2 classes of promotors intermediate element (IE) boxA boxC 2 classes: start point BoxA-IE-BoxC: Internal control region (ICR) i) INTERNAL boxA boxB ii) EXTERNAL Distal (DSE) and proximal sequence element (PSE) ( ): not all type 1 and type 2 promoters contain a TATA element 23 S. Mendo| BM24_25  Interactions at internal promotors (types 1 and 2) ‣ TFIIIA and TFIIIC are assembly factors (proteins that assist but are not part of the structure) bind to the consensus sequences and enable TFIIIB to bind at the start point. ‣ TFIIIB contains a TATA binding protein (TBP); is the only true initiation factor Internal Type 1 promotor Internal Type 2 promotor TFIIIA must bind to BoxA to enable TFIIIC bind BoxC 24 S. Mendo| BM24_25  Interactions at external promotors (type 3) ‣ TATA box can be enough to start initiation, however, ef ciency of transcription is greatly increased by: ✓ Proximal sequence element (PSE) ✓ OCT (8 bp binding sequence)/DSE element Transcription factors bind to promotor before RNA Pol III, forming a pre-initiation complex, thus it is clear that RNA Pol III does not itself recognise the promotor 25 S. Mendo| BM24_25 fi  The start point for RNA polymerase II: core promoter ‣ Responsible for the transcription of all protein coding genes, miRNAs, snRNAs (U1- U7, except U6) ✓requires: general TF (TFIIX) and activators ✓ Like bacterial promotors, short homologous sequences are present spanning from -40 to +40 around TSS +1 ✓Core promoter: minimal DNA sequence that directs accurate initiation of transcription. Categories of Polymerase II promotors Focused (found in highly regulated genes- development/di erentiation) ✓ Starting point is anked by pyrimidines (Y): surrounding CA ✓ Possess an initiator (Inr) motif between -3 and -5 at the TSS ✓ TATA box may or may not (TATA-less promotors) be present ✓ If TATA is not present, a Downstream Promotor Element (DPE) at +18 to +27 cooperatively works with Inr ✓ Includes the Inr combined with either a TATA box or a DPE ✓ Correct spacing between DPE and Inr required for optimal transcription (50% or more of promotors) Dispersed (in vertebrates 2/3 of the promotors; located in CpG islands; housekeeping genes) Have multiple (weak) TSS, distributed over 50-100 nt S. Mendo| BM24_25 fl ff  Focused vs dispersed promotors Vongoc et al. 2017 27 S. Mendo| BM24_25  Other core promoter elements ✓ In focused promotors without TATA box, other core promotor elements are present that act cooperatively with the Inr MTE motif ten element MTE -18 to -27 BRE, can be BREU, BRED of TATA 28 S. Mendo| BM24_25  Chromatin remodelling exposes the promotor ‣ Before transcription initiation can begin chromatin remodelling occurs and any nucleosome octamer positioned at the promoter has to be moved/removed. ‣ The acetylation of histones by HATs results in a dispersed structure of chromatin, which becomes accessible by TFs HATs: Histone Acetyl Transferases 29 S. Mendo| BM24_25 TATA binding proteins (TBP): a nearly universal factor ‣ TBP is a component of the positioning factor (interaction with RNA Pol) required by each Pol ( I, II and III) to bind its promotor (at the minor grove of DNA) ✓ Binds directly to TATA box if present ✓Required in most promotors whether TATA is present or absent (TATA less promotors use Inr). ✓Is present in selectivity factor SL1 (for RNA Pol I) and TFIIIB for RNA Pol III. ✓TFIID is the positioning factor for RNA Pol II (TBP+11subunits- TBP associated factors (TAFs) 30 S. Mendo| BM24_25  TATA-less pre-initiation complex ‣ Tend to be found in two classes of genes: housekeeping genes developmentally regulated genes (e.g.: development of immune system in mammals) ‣ Show similar TFIID dependence ‣ Inr when present can provide the positioning element ‣ Other TAFs in TFIID recognise the DPE element ‣ Positioning of TBP is similar to that at internal promotors of RNA Pol III 31 S. Mendo| BM24_25  Gene promotors and chromatin categories Inactive gene Poised gene: requires Active gene: requires a second signal basal TF 32 S. Mendo| BM24_25  Transcription basal apparatus assembles at the promoter The rst general TF to associate with template DNA is TFIID ‣ Ef ciency and speci city of promoter recognition TFIID: TBP+ TAFs depends on activators ‣ Some active promotors have transcripts generated upstream of the promoter (PROMPTS), e.g.: ncRNAs ‣ UPEs and the factors that bind to them increase the frequency of initiation. ‣ Binding of TFIID to the TATA box or Inr is the rst step in initiation ‣ Once RNA pol II binds to the complex: transcription initiation requires further TFs, for transcription to start. Adapted from Maxon, M. E., Goodrich, J. A., and Tijian, R. (1994). Genes Dev. 8, 515–524. 33 S. Mendo| BM24_25 fi fi fi fi  Transcription basal apparatus assembles at the promoter ‣ TFIIE and TFIIH act at later stages of initiation ‣ TFIIH is the most complex TF, as it functions both as TF and in DNA repair. Its activities include: ✓ ATPase ✓ helicases of both polarities ✓ Kinase activity (phosphorylate the CTD tail of RNA pol II: required for pol II release and progress to elongation) ✓ Interacts downstream of the start point required for RNA Pol to escape from promotor ✓ Acts at elongation ✓ Involved in DNA damage repair Adapted from Maxon, M. E., Goodrich, J. A., and Tijian, R. (1994). Genes Dev. 8, 515–524. 34 S. Mendo| BM24_25  Mediator: a molecular drawbridge ‣ Multisubunit complex of about 25-30 proteins ‣ It has a conserved region that interacts with CTD of pol II, and protein subunits that interact with the TFs at regions distant from the core promotor. Regulatory cis-acting sequences Gene-speci c TFs ‣ Connects transcriptional activators bound at enhancers or other regulatory elements with pol II. Target gene ‣ Coordinates the assembly of PIC - pre initiation complex (Pol II + general TFs at the CORE PROMOTOR core promoters to facilitate target gene transcription) 35 S. Mendo| BM24_25 fi  Initiation of transcription ‣ Multistage process that includes: Promotor melting Abortive initiation: pol synthesizes short transcripts (~7 nt) Positive transcription elongation factor-b (P- but doesn´t escape promotor TEFb) Promotor escape (>10 nt) Pausing Pause release Pol II pausing by negative P-TEFb) mediates the release of elongation factor (NELF) + paused Pol II by phosphorylating DRB-sensitivity-inducing NELF, DSIF and the CTD of Pol II; factor (DSIF) after phosphorylation DSIF becomes a positive EF. Transition to a productive elongation requires 5´capping of the RNA 36 S. Mendo| BM24_25  Promoter clearance and elongation ‣ Binding of RNA pol generates a closed complex that progresses to an open complex where DNA strands are separated. ‣ Promotor clearance is a key step in determining if a poised or an active gene is transcribed. In uenced by enhancers. 37 S. Mendo| BM24_25 fl  The C-terminal domain (CTD) of RNA pol II ‣ CDT facilities caping and splicing ‣ Throughout evolution RNA pol II acquired a repetitive structure at its terminal domain (CTD) ‣ 52 repeats of YSPTSPS (heptad repeat: 7aa); ‣ Links transcription and RNA processing ‣ Co-transcriptional surveillance ‣ Phosphorylation of CTD is required to release Pol II from the promotor (transition to elongation) ‣ Serines (S) are major sites for phosphorylation the 5th for transcription initiation; the 2nd for transcription elongation (processive) ‣ The rst phosphorylation event is catalysed by TFIIH ‣ Phosphorylation controls the movement of the RNA pol II ‣ Transcription follows 38 S. Mendo| BM24_25 fi  Transcription elongation through the nucleosome barrier https://doi.org/10.1016/j.sbi.2019.10.007 ‣ Within the cell, nucleosomes present a challenging barrier to elongating polymerases, leading to pausing or backtracking ‣ Nucleosomes are disrupted on active RNA pol II transcribed genes by H2A- H2B removal, aided by auxiliary elongation factors (EFs): FACT (facilitates chromatin transcription), Elongator, and TFIIS 39 S. Mendo| BM24_25  Co-transcriptional processing ‣ Transcription and post-transcription are connected through multifunctional proteins ‣ While transcription is occurring at the 5´ end, the pre-mRNA is immediately modi ed (as the 1st 25 nt emerge from the exit channel of RNA pol II) by the addition of 5´cap (7-methyl guanosine cap - m7G) ‣ Capping enzyme complex (CEC) is bound to RNA pol II before transcription starts, so RNA pol II transcripts are 5´ capped immediately 40 S. Mendo| BM24_25 The cap-binding complex ‣ Cap is bound to cap-binding complex (CBC) ‣ CBC is recognised by the nuclear pore ‣ Facilitating the export from the nucleus, protecting mRNA from decapping enzymes ‣ CBC is replaced by eIF4E 41 S. Mendo| BM24_25  Nuclear import and export ‣ Small polar molecules, ions and macromolecules (proteins and RNAs) are transported by an active process (not passive diffusion) 42 S. Mendo| BM24_25  The Ran cycle ‣ Ran plays a key role in the import and export of molecules in and out of the nucleus Ran GAP: GTP activating proteins GEF: Guanine nt exchange factor Ran 43 S. Mendo| BM24_25  Regulated nuclear import and export of TFs and proteins ‣ Regulatory proteins must be delivered to their site of activity in the nucleus ‣ Traf cking occurs via nuclear pore complexes (NPCs), which allow bidirectional passive diffusion of small molecules ‣ Proteins targeted to the nucleus have a speci c (aa) sequence: nuclear localization signal (NLS) ‣ Other nuclear proteins shuttle between the nucleus an cytoplasm, and require a nuclear export signal (NES) ‣ Nuclear import and export pathways (active transport) are mediated by soluble receptors known as importins and exportins (karyopherins family) 44 S. Mendo| BM24_25 fi fi Nuclear import and export: TFs, Proteins (e.g.:DNA repair) 45 S. Mendo| BM24_25 Regulated nuclear import and export of TFs and proteins 46 S. Mendo| BM24_25  Termination of Transcription. The 3´ ends of mRNA ‣ Required to recycle RNA pol II ‣ Triggered by recognition of the Poly(A) site ‣ The sequence AAUAAA is a signal for cleavage to generate a 3′ end of mRNA that is polyadenylated. ‣ The reaction requires a protein complex that contains i) a speci city factor, ii) an endonuclease, and iii) poly(A) polymerase (PAP). ‣ The speci city factor and endonuclease cleave RNA downstream of AAUAAA. ‣ The speci city factor and PAP add about 200 A residues processively to the 3′ end. ‣ The poly(A) tail controls mRNA stability and in uences translation. De nition of the site of cleavage/polyadenilation: ‣ upstream AAUAAA motif ‣ U / UG rich element ✓ Can occur many nt after the STOP codon ✓ De ne the 3ÚTR 47 S. Mendo| BM24_25 fi fi fi fi fl  Cleavage and poly-A at the 3´end ✓3´ processing complex consists of several activities ‣ CPSF and CstF bind to speci c regions ‣ PAP adds a short oligo(A) sequence ~10 residues at 3´ (slowly) ‣ Nuclear poly(A) binding protein (PABPII) binds to the polyA tail allows extension to ~200 A´s by PAP. ‣ Mature mRNA remains bound by Poly(A) to PABI protecting it from degradation, but binds also to eIF4G to facilitate translation. Cleavage and polyadenylation specify factor (CPSF) Cleavage stimulation factor (CstF) ‣ Avoid degradation ✓ Cleavage is performed by cleavage factors I and II (CFI and CFII) ‣ promote nuclear export ‣ promote translation 48 S. Mendo| BM24_25 fi  Histone mRNA 3´end formation 100 kDa zinc nger processing factor (ZFP100) Molecular adapter interacting with different components cleavage site SLBP (HBP) stem loop BP: coordinate ‣ formation of 3´end ‣ translation in histone proteins ‣ degradation of H mRNA ‣ Do not contain introns; do not end with a poly (A) tail ‣ End with a conserved stemloop element followed by a purine rich histone downstream element (HDE) ‣ HDE interacts with U7 snRNP (one of the snRNP not involved in splicing) ‣ As it does not have a Poly(A), stimulation of translation is in charge of proteins (SLBP) ‣ Histone mRNA are high during S-phase (DNA synthesis) as DNA must be packaged by histones ‣ Mediated by a nuclease cleavage/polyadenylation speci city factor- CPSF (endonuclease and exonucleolytic activity) that interacts with U7 49 S. Mendo| BM24_25 fi fi  Summary: Initiation is followed by promotor clearance and elongation guanylyl transferase (adds G to In the PIC ser of CTD are not 5´mRNA-capping) binds phosphorylated to CTD phosphorylated at Ser5 RNA pol is non-processive ✓ Promotor clearance: key regulated step in uenced by enhancers, determining if a poised or active gene will be transcribed (PIC) ✓ Phosphorylation of the CTD tail at serine 5 is required for the transition to elongation ✓ CTD phosphorylation at serine 2 occurs during elongation and is involved in release from pausing ✓The CTD coordinates processing and export of RNA from the nucleus with transcription CTD connects other processes with transcription The Paf1 (polymerase associated factor 1) complex regulates when or if a gene is transcribed. Therefore, works with the CTD to coordinate co- and post- https://www.youtube.com/watch?v=XzVXhemtwmA (processo completo) transcriptional transcript processing and export Reprinted from Gardner, P. P., and Vinther, J. (2008). Mutation of miRNA target sequences. Trends Genet. 24, 262–265. ©2008, with permission from Elsevier 50 (http://www.sciencedirect.com/science/journal/01689525). S. Mendo| BM24_25 fl  Co-transcriptional processing revisited 1. Pol II with the hypophosphorylated CTD is assembled into the preinitiation complex (PIC) at the promoter 2. Ser5 residues of the CTD heptapeptide repeats are phosphorylated 3. Pol II is stalled by the concerted actions of two negative elongation factors, DSIF and NELF (inhibitory protein complexes-check point) 4. Facilitates the recruitment of capping enzymes to ensure proper capping of the nascent pre-mRNA 5. P-TEFb phosphorylates DISF, NELF, and the CTD repeats at the Ser2 positions. 6. These phosphorylation events promote the dissociation of NELF and convert DSIF into a positive EF, allowing the Pol II to engage in productive elongation to produce full-length transcripts. 51 S. Mendo| BM24_25  SUMMARY ‣ Each RNA polymerase transcribe speci c genes. ‣ They do not recognise their promotors directly. ‣ TFs recognise characteristic sequence elements of any particular promoter and assist on positioning RNA polymerase at the correct TSS. ‣ The factor TBP is required for initiation by all RNA polymerases. ‣ RNA pol II can be focused (short sequence elements in the region upstream of the promoter) or dispersed (numerous weak TSS); the latter found in CpG islands. ‣ TATA box may or may not be present; if present TBP binds directly to it. ‣ In TATA-less promoters TBP binds to Inr and DPE. ‣ The CTD of RNA pol II is phosphorylated during initiation; it provides a point of contact for other proteins that modify the transcript (5´capping, splicing factors, 3´processing complex and mRNA export from the nucleus. ‣ Termination capacity of Pol II is linked to 3´ end formation of mRNA. ‣ Promotors may be stimulated by enhancers, which act at great distances in either orientation on either side of the gene. ‣ Enhancers can assemble protein complexes that interact with proteins bound at the promoter, requiring that DNA is looped out, and can recruit complexes that modify chromatin structure. 52 S. Mendo| BM24_25 fi