Gene reporter.

Presentazione sul tema: "Gene reporter."— Transcript della presentazione:

1 Gene reporter

2 Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

3 I promotori eucariotici sono costituiti
da una associazione variabile di “box” 1 -20 -40 -60 -80 -100 10 -120 -140 Ottamero CAAT GC SV40 (promotore precoce) Timidina kinasi Istone H2B

4 Attivatori costitutivi
Modulo Attivatore DNA occupato Consenso Distribuzione CAAT box CTF/NF1 22 bp GGCCAATCT ubiquitaria GC box SP1 20 bp GGGCGG Ottamero Oct-1 ATTGCAT Oct-2 23 bp linfoidi kB NFkB 10 bp GGGACTTTCC ATF GTGACGT

5 Attivatori inducibili e elementi di risposta
Agente regolatore Elemento di risposta Consenso Fattore Dimensioni (dalton) Shock termico HSE CNNGCCNNTCCNNG HSTF 93.000 Glucocorticoidi GRE TGGTACAAATGTTCT GR 94.000 Cadmio MRE CGNCCCGGNCNC TPA TRE TGACTCA AP1 39.000 Siero SRE CCATATTAGG SRF 52.000

6 Esempio di promotore

7 Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

8 Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

9 Enhancers Similar sequence elements are found in enhancers and promoters. Enhancers form complexes of transcription factors that interact directly or indirectly with the promoter.

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16 Ruolo degli Isolatori

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18 Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

19 Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

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21 LCR and insulators An LCR is located at the 5' end of the domain and consists of several hypersensitive sites. Insulators are specialized chromatin structures that have hypersensitive sites. All known insulators are able to block passage of any activating or inactivating effects from enhancers, silencers, or LCRs. In some cases, insulators have directionality, and may stop passage of effects in one direction but not the other.

22 Fattori di trascrizione negli eucarioti
(trans-acting factors = fattori che agiscono in trans ) Possono essere distinti in: - Fattori generali - Fattori specifici (attivatori)

23 Complesso di inizio su un promotore Pol II Pol Pol II 1 TBP + vari TAF
TATA -1 -2 -3 -4 -5 1 2 TFII D TFII A TFII B Pol II TFII E 1 TBP + vari TAF TFIIF

24 Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

25 Lewin, IL GENE VIII, Zanichelli editore S.p.A. Copyright © 2006

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27 Attivazione della trascrizione

28 Complessi basali delle RNA polimerasi eucariotiche
Figure 1 | Basal transcription machineries and promoter structures of the eukaryotic DNA-dependent RNA polymerases I, II and III. a | Assembly of the RNA polymerase (Pol) I pre-initiation complex (PIC) at ribosomal RNA gene (rDNA) promoters begins with the binding of upstream binding factor (UBF) to the upstream control elements (UCEs) and core element of the rDNA promoter, leading to the recruitment of the SL‑1 initiation factor, which contains TATA-box-binding protein (TBP) and at least five TATA-box-associated factors (TAFs). The resultant stable UBF–SL‑1 complex recruits an initiation-competent form of RNA Pol I, which contains RRN3 that mediates interactions between RNA Pol I and SL‑1 (REF. 189). b | For RNA Pol II transcription, TBP initiates PIC assembly by binding to the TATA box at the promoter. TFIIA and TFIIB interact with TBP and reinforce its binding to DNA. In turn, TFIIB recruits RNA Pol II and TFIIF, thus positioning RNA Pol II over the transcription start site (TSS). TFIIH mediates ‘melting’ of the TSS to form the open complex that is stabilized by TFIIE190. The dashed outline of TBP indicates that it is part of the TFIID complex. c | RNA Pol III PICs differ in composition depending on the class of genes transcribed. Most RNA Pol III-transcribed genes (for example, those that encode tRNAs) have internal promoters that comprise two sequence blocks (A and B) that are located in the transcribed region. The A and B blocks are recognized by TFIIIC that recruits TFIIIB, which is composed of the subunits B‑related factor 1 (BRF1), BDP1 and TBP. Finally, TFIIIB recruits RNA Pol III. For 5S rDNA promoters the B block is replaced by a sequence, termed block C, to which TFIIIA binds and recruits and orientates TFIIIB, following which transcription initiation proceeds as for tRNA genes. For a small number of RNA Pol III-transcribed genes (for example, U6 snRNA (R?N?U?6?‑1?)) the promoters are located upstream of the gene and contain TATA boxes bound by TBP, and proximal sequence elements (PSEs) bound by a complex called small nuclear RNA-activating protein complex (SNAPC). These upstream promoters are bound by a different form of TFIIIB from tRNA genes, which is composed of BRF2, BDP1 and TBP9.

29 Initiation at pol II promoters
Binding of TFIID to the TATA box is the first step in initiation. Other transcription factors bind to the complex in a defined order, extending the length of the protected region on DNA. When RNA polymerase II binds to the complex, it initiates transcription.

30 Later events of pol II initiation
TFIIE and TFIIH are required to melt DNA to allow polymerase movement. Phosphorylation of the CTD may be required for elongation to begin. Further phosphorylation of the CTD is required at some promoters to end abortive initiation. The CTD may coordinate processing of RNA with transcription.

31 Fattori di trascrizione negli eucarioti
(trans-acting factors = fattori che agiscono in trans ) Possono essere distinti in: - Fattori generali - Fattori specifici (attivatori)

32 Struttura“modulare” degli attivatori trascrizionali
legame al DNA transattivazione dimerizzazione legame al ligando

33 Strategia per lo studio degli attivatori

34 Struttura degli attivatori

35 Domini (moduli) degli attivatori

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37 Scambio di domini (attivatori chimerici)

38 Scambio di domini N C modulatore DNA ligando hGR hER Recettore “chimera”

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40 Combinazioni di omodimeri ed eterodimeri

41 Domini di dimerizzazione

42 Domini di attivazione Domini acidici: es. GAL4
Domini ricchi di glutammine: es. Sp1 Domini ricchi di proline: es. CTF

43 Famiglie di fattori di trascrizione
Domini a zinco Omeodominio (elica-giro-elica) HTH Elica-ansa-elica HLH Cerniera di leucina

44 Coattivatori della trascrizione

45 Coattivatori della trascrizione

46 Complesso del mediatore
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

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49 Complesso d’inizio di pol II

50 Modelli dell'oloenzima

51 RNA pol II holoenzyme Includes pol II, GTFs, Srb/Mediator complex
Exists in more than one form Can initiate transcription and respond to activators

52 Regolazione negativa

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54 Regolazione degli attivatori

55 Trasduzione del segnale dalla membrana al nucleo

56 Le vie delle MAP chinasi

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58 Trasduzione del segnale dalla membrana al nucleo