Il differenziamento cellulare dipende da meccanismi di regolazione dell espressione genica

Transcription

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3 Il differenziamento cellulare dipende da meccanismi di regolazione dell espressione genica

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9 RNA Ribonucleotidi monofosfato uniti a formare una catena polinucleotidica

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11 I precursori della sintesi sono i ribonucleotidi trifosfato. L energia che occorre per la formazione del legame fosfodiesterico è data dall eliminazione del pirofosfato per idrolisi del legame.

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13 L enzima che catalizza l unione dei ribonucleotidi è l RNA polimerasi

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15 The human RNA polymerases Polymerase Location Product RNA polymerase I nucleolus 18S, 28S, 5.8S rrna RNA polymerase II nucleoplasm hnrna/mrna, U1, U2, U4, U5 snrna RNA polymerase III nucleoplasm mitochondrial RNA polymerase trna, 5S RNA, U6 snrna, 7SL RNA mitochondrion all mitochondrial RNA

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17 b). Gene structure promoter region exons (filled and unfilled boxed regions) +1 introns (between exons) transcribed region mrna structure 5 3 translated region

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19 Structure of eukaryotic mrna 5 Cap 7mGppp 5 untranslated region initiation AUG translated region 3 untranslated region UGA termination polyadenylation signal AAUAAA (A) ~200 poly(a) tail all mrnas have a 5 cap and all mrnas (with the exception of the histone mrnas) contain a poly(a) tail the 5 cap and 3 poly(a) tail prevent mrna degradation loss of the cap and poly(a) tail results in mrna degradation 3

20 closed promoter complex Transcription RNA polymerase open promoter complex initiation elongation termination RNA product

21 Legame al promotore della RNA polimerasi Apertura della doppia elica Inizio della sintesi Allungamento Terminazione

22 Direzione della sintesi Filamento senso Filamento antisenso

23 5....AGAAGATG ATGTCGGGCCAAACGCTCACGGATCGGATCGCCGCCGCTCAGTACAGCGTTACAGGCTCTGCTGT AGCAAGAGCGGTCTGCAAAGCCACTACTCATGAAGTAATGGGCCCCAAGAAAAAGCACCTGGACTATTTGATCCAGGC TACCAACGAGACCAATGTTAATATTCCTCAGATGGCCGACACTCTCTTTGAGCGGGCAACAAACAGTAGCTGGGTGGTT GTGTTTAAGGCTTTAGTGACAACACATCATCTCATGGTGCATGGAAATGAGAGATTTATTCAATATTTGGCTTCTAGAAA TACACTATTCAATCTCAGCAATTTTTTGGACAAAAGTGGATCCCATGGTTATGATATGTCTACCTTCATAAGGCGCTATA GTAGATATTTGAATGAAAAGGCTTTTTCTTACAGACAGATGGCCTTTGATTTTGCCAGGGTGAAGAAAGGGGCCGATGG TGTAATGAGGACAATGGCTCCCGAAAAGCTGCTAAAGAGTATGCCAATACTACAGGGACAAATTGATGCACTGCTTGAA TTTGATGTGCATCCAAATGAACTAACAAATGGTGTCATAAATGCAGCATTTATGCTTCTTTTCAAAGATCTTATCAAACTT TTTGCTTGCTACAATGATGGTGTTATTAACTTACTCGAAAAGTTTTTTGAAATGAAGAAAGGACAATGTAAAGATGCTCTA GAAATTTACAAACGATTTCTAACTAGAATGACACGAGTGTCTGAATTTCTCAAGGTTGCAGAGCAAGTTGGTATTGATAA AGGTGACATTCCTGACCTCACACAGGCTCCCAGCAGTCTTATGGAGACGCTTGAACAGCATCTAAATACATTAGAAGGA AAGAAACCTGGAAACAATGAAGGATCTGGTGCTCCCTCTCCATTAAGTAAGTCTTCTCCAGCCACAACTGTTACGTCTC CTAATTCTACACCAGCTAAAACTATTGACACATCCCCACCGGTTGATTTATTTGCAACTGCATCTGCGGCTGTCCCAGTC AGCACTTCTAAACCATCTAGTGATCTCCTGGACCTCCAGCCAGACTTTTCCTCTGGAGGGGCAGCAGCAGCCGCAGCA CCAGCACCACCACCACCTGCTGGAGGAGCCACTGCATGGGGAGACCTTTTGGGAGAGGATTCTTTGGCTGCACTTTCC TCTGTTCCCTCTGAAGCACAGATTTCAGATCCATTTGCACCAGAACCTACCCCTCCTACTACAACTGCTGAAATTGCAAC CACTACTGCTGCCACCGCCGCTGCCACCACCACTACCATTCATCTCTTGCCAGCTTAGTAGGCAATCTTGGAATTTCTG GTACCACAACAAAAAAGGGAGATCTTCAGTGGAATGCTGGAGAGAAAAAGTTGACTGGTGGAGCCAACTGGCAGCCTA AAGTAGCTCCAGCAACCTGGTCAGCAGGCGTTCCACCAAGTGCACCTTTGCAAGGAGCTGTACCTCCAACCAGTTCAG TTCCTCCTGTTGCCGGGGCCCCATCGGTTGGACAACCTGGAGCAGGATTTGGAATGCCTCCTGCTGGGACAGGCATG CCCATGATGCCTCAGCAGCCGGTCATGTTTGCACAGCCCATGATGAGGCCCCCCTTTGGAGCTGCCGCTGTACCTGGC ACGCAGCTTTCTCCAAGCCCTACACCTGCCAGTCAGAGTCCCAAGAAACCTCCAGCAAAGGACCCATTAGCGGATCTTA ACATCAAGGATTTCTTGTAAACAATTTAAGCTGCAATATTTGTGACTGAATAGGAAAATAAATGAGTTTGGAGACTTCAAA TAAGATTGATGCTGAGTTTCAAAGGGAGCCACCAGTACCAAACCCAATACTTACTCATAACTTCTCTTCCAAAATGTGTA ACACAGCCGTGAAAGTGAACATTAGGAATATGTACTACCTTAGCTGTTATCCCTACTCTTGAAATTGTAGTGTATTTGGA TTATTTGTGTATTGTACGATGTAAACAATGAATGGATGTTACTGATGCCGTTAGTGCTTTTTTGGACTTCACCTGAGGAC AGATGATGCAGCTGTTGTGTGGCGAGCTATTTGGAAAGACGTCTGTGTTTTTGAAGGTTTCAATGTACATATAACTTTTG AACAAACCCCAAACTCTTCCCATAAATTATCTTTTCTTCTGTATCTCTGTTACAAGCGTAGTGTGATAATACCAGATAATA AGGAAAACACTCATAAATATACAAAACTTTTTCAGTGTGGAGTACATTTTTCCAATCACAGGAACTTCAACTGTTGTGAGA AATGTTTATTTTTGTGGCACTGTATATGTTAA..3..3

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26 Sequence elements within a typical eukaryotic gene 1 1 based on the thymidine kinase gene octamer transcription element promoter +1 ATTTGCAT GC CAAT GC TATA TATA box (TATAAAA) located approximately bp upstream of the +1 start site determines the exact start site (not in all promoters) binds the TATA binding protein (TBP) which is a subunit of TFIID GC box (CCGCCC) binds Sp1 (Specificity factor 1) CAAT box (GGCCAATCT) binds CTF (CAAT box transcription factor) Octamer (ATTTGCAT) binds OTF (Octamer transcription factor)

27 Proteins regulating eukaryotic mrna synthesis General transcription factors TFIID (a multisubunit protein) binds to the TATA box to begin the assembly of the transcription apparatus the TATA binding protein (TBP) directly binds the TATA box TBP associated factors (TAFs) bind to TBP TFIIA, TFIIB, TFIIE, TFIIF, TFIIH 1, TFIIJ assemble with TFIID RNA polymerase II binds the promoter region via the TFII s Transcription factors binding to other promoter elements and transcription elements interact with proteins at the promoter and further stabilize (or inhibit) formation of a functional preinitiation complex 1 TFIIH is also involved in phosphorylation of RNA polymerase II, DNA repair (Cockayne syndrome mutations), and cell cycle regulation

28 17q11.1-q TAF15 1p TAF13 1p TAF12 6p TAF11 11p TAF10 5q11.2-5q TAF9 6p TAF8 Xq TAF7L 5q31 55 TAF7 11q12.3 TAF6L 7q TAF6 10q24-10q TAF5 18q TAF4B 20q TAF4 10p TAF3 8q TAF2 9p like TAF1L Xq TAF1 Chromosoma Alias Nome TF2D TBP + TAF

29 Binding of the general transcription factors E F TAFs B TFIID H A TBP J TFIID (a multisubunit protein) binds to the TATA box to begin the assembly of the transcription apparatus the TATA binding protein (TBP) directly binds the TATA box TBP associated factors (TAFs) bind to TBP TFIIA, TFIIB, TFIIE, TFIIF, TFIIH, TFIIJ assemble with TFIID

30 Binding of RNA polymerase II E F B TFIID H A TBP J RNA pol II RNA polymerase II (a multisubunit protein) binds to the promoter region by interacting with the TFII s TFs recruit histone acetylase to the promoter

31 TATA BOX BINDING PROTEIN TBP Saddle-like domain TATA BOX DNA BINDING

32 TAF5 stabilizes TAFs interaction, specially histonelike ones (TAF6, TAF9) TAF1: Acetyl transferase activity Interaction with TFIIF TAF6 TAF11 TAF4 TAF12 TAF9 TAF13 TAF3 TAF12 TAF8 TAF4 TAF10 TBP TAF7 TAF5 TAF5 TAF11 TAF8 TAF3 TATA BOX TAF13 TAF6 TAF9TAF10 TAF1

33 TAF12 TAF4 TAF1 TAF4 TAF6 TAF11 TAF5 TAF8 TAF10 TAF7 TBP TAF5 TAF11 TAF8 TAF3 TATA BOX TAF13 TAF6 TAF9TAF10 TAF9 TAF13 TAF12 TAF3 DNA BENDING

34 TAF12 TAF4 TAF5TFIID TAF1 TAF8 TAF10TBP TAF7 TAF5 TAF11 TAF8 TAF3 TATA BOX TAF13 TAF6 TAF9TAF10 TAF6 TAF11 TAF4 TAF9 TAF13 TAF12 TAF3

35 HETEROTRIMER (A, B, C SUBUNITS) BINDS PROMOTER REGION WITH TFIID TFIIA DAB TFIID TFIIB TWO DOMAIN: N-TERMINAL Zn-RIBBON AND RNA POL II TFIIF CORE DOMAIN HETEROTETRAMER (RAP30) 2 (RAP74) 2 (RAP30) 2 BINDS RNA POL II AND TFIIB. RAP74 INTERACTS WITH DNA. TFIIF STABILIZES THE INTERACTION OF RNA POLII WITH PROMOTER AND STIMULATES ELONGATION RATE TFIIE TFIIH TFIIB INTERACTS WITH SADDLE-LIKE DOMAIN OF TBP AND WITH BENDED DNA ON SIDES OF TATA BOX. TBP/TFIIB COMPLEX RECRUITS RNA POLII AND OTHER GTF P TFIIE MODULATES THE HELICASE AND KINASE ACTIVITIESOF TFIIH BY STIMULATING CTD DOMAIN RNA POLII PHOSPHORYLATION TFIIH STIMULATES PROMOTER MELTING AND IT PHOSPHORYLATION OF HAS KINASE RNA POLII CTD STIMULATES ACTIVITY AGAINST PROMOTER MELTING AND RNA POL II TRANSCRIPTION START

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37 DAB RNA POLII WITH PROMOTER AND STIMULATES ELONGATION RATE TWO DOMAIN: HETEROTRIMER (A, B, C SUBUNITS) BINDS PROMOTER REGION WITH TFIID TFIIA TFIID TFIIB N-TERMINAL Zn-RIBBON AND RNA POL II TFIIF CORE DOMAIN TFIIE TFIIH MODULA LE ATTI- VITA ELICASICA TFIIB INTERACTS WITH SADDLE-LIKE DOMAIN OF TBP AND WITH BENDED DNA ON SIDES OF TATA BOX. TBP/TFIIB COMPLEX RECRUITS RNA POLII AND OTHER GTF E UN ETEROTETRAMERO (RAP30) 2 (RAP74) 2. RAP30 LEGA RNA POLII E TFIIB; RAP74 INTERAG. CON IL DNA (5bp A MONTE DEL TS STABILIZZA L INTERAZIONE DI RNA POLII CON IL PROMOTER. STIMOLA TASSO DI ELONGATION E CHINASICA DI TFIIH (STIMOLAN- DO LA FOSFORI- LAZIONE DEL CTD-DOMAIN DEL- LA RNA POLII). CONSTA DI DUE SUBUNITA

38 Steps in mrna processing (hnrna is the precursor of mrna) capping (occurs co-transcriptionally) cleavage and polyadenylation (forms the 3 end) splicing (occurs in the nucleus prior to transport) exon 1 intron 1 exon 2 cap Transcription of pre-mrna and capping at the 5 end Cleavage of the 3 end and polyadenylation cap cap poly(a) Splicing to remove intron sequences cap poly(a) Transport of mature mrna to the cytoplasm

39 Polyadenylation cleavage of the primary transcript occurs approximately nucleotides 3 -ward of the AAUAAA consensus site polyadenylation catalyzed by poly(a) polymerase approximately 200 adenylate residues are added cleavage AAUAAA mgpppnmpnm mgpppnmpnm AAUAAA A A A polyadenylation A A A 3 poly(a) is associated with poly(a) binding protein (PBP) function of poly(a) tail is to stabilize mrna

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45 Splicing Rimozione di un introne attraverso due reazioni sequenziali di trasferimento di fosfato, note come transesterificazioni. Queste uniscono due esoni rimuovendo l introne come un cappio

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47 EXON SHUFFLING

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50 GENE PREDICTION GeneScan GrailEXP Promoter 2.0 Omiga 2.0 GeneMark F GENE SH

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52 Chemistry of mrna splicing two cleavage-ligation reactions transesterification reactions - exchange of one phosphodiester bond for another - not catalyzed by traditional enzymes branch site adenosine forms 2, 5 phosphodiester bond with guanosine at 5 end of intron intron 1 Pre-mRNA 2 OH-A branch site adenosine exon 1 exon 2 5 G-p-G-U - A-G-p-G 3 First clevage-ligation (transesterification) reaction

53 ligation of exons releases lariat RNA (intron) intron 1 U-G-5 -p-2 -AA Splicing intermediate exon 1 exon 2 5 G-OH 3 A-G-p-GA - 3 Second clevage-ligation reaction intron 1 Lariat U-G-5 -p-2 -A Spliced mrna 3 G-A exon 1 exon 2 5 G-p-G 3

54 Recognition of splice sites invariant GU and AG dinucleotides at intron ends donor (upstream) and acceptor (downstream) splice sites are within conserved consensus sequences donor (5 ) splice site branch site acceptor (3 ) splice site G/GUAAGU... A... YYYYYNYAG/G U1 U2 small nuclear RNA (snrna) U1 recognizes the donor splice site sequence (base-pairing interaction) U2 snrna binds to the branch site (base-pairing interaction) Y= U or C for pyrimidine; N= any nucleotide

55 intron 1 Step 2: binding of U4, U5, U6 2 OH-A exon 1 exon 2 U5 5 G-p-G-U - A-G-p-G 3 U1 U2 U4 U6 intron 1 Step 3: U1 is released, then U4 is released 2 OH-A exon 1 exon 2 U5 U6 U2 5 G-p-G-U - A-G-p-G 3

56 Step 4: U6 binds the 5 splice site and the two splicing reactions occur, catalyzed by U2 and U6 snrnps intron 1 mrna 3 G-A U6 2 OH-A U-G-5 -p-2 -A U5 U2 5 G-p-G 3

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60 Trans-splicing Nei protozoi e in un nematode

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63 Differenti molecole di mrna dallo stesso gene Splicing alternativo Uso di promotori alternativi Uso di segnali di poliadenilazione alternativi

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68 Structure of prokaryotic messenger RNA 5 Shine-Dalgarno sequence PuPuPuPuPuPuPuPu 3 AAU termination translated region initiation AUG The Shine-Dalgarno (SD) sequence base-pairs with a pyrimidine-rich sequence in 16S rrna to facilitate the initiation of protein synthesis

69 Il gene dei procarioti è policistronico

70 Enhancers Nei geni degli eucarioti gli enhancers possono distare dalla regione codificante anche più di 50 Kb.

71 Regolazione dell espressione genica Organizzazione della cromatina Punto 1 Inizio della trascrizione

72 Meccanismi di Regolazione dell espressione genica Fase Nucleare Scelta del gene che deve essere espresso Maturazione dell RNA Trasferimento Nucleo Citoplasma Fase Citoplasmatica Sintesi delle catene polipeptidiche Modificazioni post-traduzionali Trasferimento delle proteine nelle sedi di competenza

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74 GENE ACTIVATOR PROTEIN Covalent histone modifications

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76 Modificazioni covalenti degli istoni