Sistemi (sperimentali) di espressione

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1 Sistemi (sperimentali) di espressione
In vitro: - estratti cellulari In vivo: - cellule procariotiche cellule eucariotiche in coltura - animali transgenici

2 Trasfezione di cellule in coltura
Vettore Tipo vettori plasmidici transiente o stabile vettori virali transiente/stabile siRNA transiente

3 Gene reporter

4 (profilo trascrizionale)
Microarray (profilo trascrizionale)

5 Profilo differenziale mediante microarray

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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

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10 RNA polimerasi eucariotiche

11 RNA polymerases RNA polymerase I synthesizes rRNA in the nucleolus.
RNA polymerase II synthesizes mRNA in the nucleoplasm. RNA polymerase III synthesizes small RNAs in the nucleoplasm. All eukaryotic RNA polymerases have ~12 subunits and are aggregates of >500 kD. Some subunits are common to all three RNA polymerases. The largest subunit in RNA polymerase II has a CTD (carboxy- terminal domain) consisting of multiple repeats of an eptamer.

12 Promotori della pol III
U6

13 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

14 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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16 I geni per gli rRNA sono ripetuti in tandem nei genomi eucariotici
ETS 18S ITS 28S NTS RNA RNA DNA DNA gene promoter 60/81 bp repeats spacer other repetitive elements DNA

17 I precursori degli rRNA sono piu' lunghi della somma degli rRNA maturi

18 rRNA genes Ribosomal RNA is coded by a large number of identical genes that are tandemly repeated to form a cluster(s). Each rDNA cluster is organized so that transcription units giving a joint precursor to the major rRNAs alternate with nontranscribed spacers.

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

20 Il complesso d'inizio della pol I
The structural organization of mammalian rDNA repeats and the basal factors required for transcription initiation. The diagram shows the arrangement of tandemly repeated rRNA genes. The site of transcription initiation of 47S pre-rRNA (black arrow) and intergenic transcripts from the spacer promoter (red arrow) are indicated. Terminator elements are located downstream of the transcription unit (T1−10), downstream of the spacer promoter (TSP), and upstream of the gene promoter (To) (red boxes). Repetitive enhancer elements (gray boxes) are located between the spacer promoter and major gene promoter. The ellipsoids show the factors that are associated with the rDNA promoter and Pol I, respectively. TTF-I is associated with the upstream terminator To. Synergistic binding of UBF and TIF-IB/SL1 to the rDNA promoter is required for the recruitment of RNA polymerase I (Pol I)—together with multiple Pol I-associated factors—to the transcription start site to initiate pre-rRNA synthesis.

21 Il complesso d'inizio della pol I
UBF: inizio, allungamento SL1: inizio TIF-1A (RRN3): inizio TTF-1: terminazione

22 Regolazione della sintesi dell'rRNA
Regulation of Pol I transcription in response to external signals. The bar diagrams show the relative levels of pre-rRNA upon exposure of cells to amino acid starvation (left), exposure to oxidative stress (middle), and growth factor stimulation (right).

23 Regolazione della sintesi dell'rRNA
Regulation of Pol I transcription during cell cycle progression. UBF is activated during interphase by phosphorylation of serine 484 (S484) by Cdk4/cyclin D and phosphorylation of serine 388 (S388) by Cdk2/cyclin E and A. At the entry into mitosis, phosphorylation of TAFI110 at threonine 852 (T852) by Cdk1/cyclin B inactivates TIF-IB/SL1. At the exit from mitosis, Cdc14B dephosphorylates T852, leading to recovery of TIF-IB/SL1 activity. Activating phosphorylations are marked in green, inhibiting ones in red.

24 Farmaci chemioterapici che inibiscono la sintesi dell'rRNA

25 Il promotore minimo della pol II
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

26 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

27 Gene reporter

28 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

29 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

30 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

31 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

32 Esempio di promotore

33 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

34 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

35 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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39 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

40 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

41 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

42 Ruolo degli Isolatori

43 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

44 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

45 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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47 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.

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

49 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

50 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

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

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53 Attivazione della trascrizione

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

55 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.

56 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.

57 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.