Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p

Presentazione sul tema: "Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p"— Transcript della presentazione:

1

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

4 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.

5 Promotori della pol III
U6

6 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

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

9 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

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

11 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.

12 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

13 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.

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

15 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).

16 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.

17 Chemioterapici inibiscono la sintesi dell'rRNA

18 Gene reporter

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 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

23 Esempi di “box” di promotori eucariotici

24 Esempi di box del tipo “elemento di risposta”
Agente regolatore Modulo Consensus Fattore Grandezza (dalton) Shock termico Glucocorticoidi Cadmio TPA Siero HSE GRE MRE TRE SRE CNNGCCNNTCCNNG TGGTACAAATGTTCT CGNCCCGGNCNC TGACTCA CCATATTAGG HSTF Recettore ? AP1 SRF 93,000 94,000 39,000 52,000

25 Esempio di promotore

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 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

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

29

30

31

32 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

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 Ruolo degli Isolatori

36 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

37 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

38 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

39

40 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.