1. Chromatin immunoprecipitation (Chip)

3. Chromatin immunoprecipitation (Chip)

4. Chip in lievito per Gal4 e Sug1
An example of a ChIP assay done in yeast using an antibodies raised against the gene-specific transcription factor Gal4 and the general transcription factor and proteasome constituent Sug1. Different PCR primers (amplifying DNA segments A-E) were employed to probe for the presence of the two proteins along the GAL1 gene under non-inducing (raffinose) or inducing (galactose) conditions. Gal4 is known to be localized to the promoter. Therefore, the ChIP signals in regions B and C for this protein reflect the presence of longer DNAs in the sample which are co-IP’d with Gal4 and amplify using the B and C region primers. This reflects the relatively low resolution of the ChIP assay. Sug1 is an elongation factor and is therefore found throughput the gene.

5. Coimmunoprecipitazione

6. Affinity co-purification
(pull down)

7. Sistema dei due ibridi

8. Sistema dei due ibridi

9. Sistema dei tre ibridi

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

12. Ricombinazione omologa

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

15. 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

16. 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

17. Vettori episomali

18. Trasfezione di cellule in coltura
vettori plasmidici
vettori virali
vettori episomali
siRNA
transiente
stabile

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. 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

21. 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

22. 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

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

26. RNA polimerasi eucariotiche

27. 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.

28. Promotori della pol IIIU6

29. 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

30. 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

32. 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

33. I precursori degli rRNA sono piu' lunghi della somma degli rRNA maturi

34. 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.

35. 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

36. 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.

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

38. 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).

39. 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.

40. Chemioterapici inibiscono la sintesi dell'rRNA

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

42. 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

43. Gene reporter

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. 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

46. 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

47. 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

48. Esempio di promotore

49. 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

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

55. 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

56. 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

57. 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

58. Ruolo degli Isolatori

59. 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

60. 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

61. 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

63. 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.

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

65. 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

66. 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

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

69. Attivazione della trascrizione

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

71. 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.

72. 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.

73. 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.