Biologia molecolare - Robert F. Weaver Copyright © 2005 – The McGraw-Hill Companies srl Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Biologia molecolare - Robert F. Weaver Copyright © 2005 – The McGraw-Hill Companies srl 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 (carboxyterminal domain) consisting of multiple repeats of an eptamer. I geni per gli rRNA sono ripetuti in tandem nei genomi eucariotici NTS ETS 18S 28S ITS RNA RNA DNA DNA DNA other repetitive elements 60/81 bp repeats spacer promoter 60/81 bp repeats spacer promoter gene promoter I PRECURSORI DEGLI rRNA SONO PIU' LUNGHI DELLA SOMMA DEI DUE rRNA MATURI 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. Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Biologia molecolare - Robert F. Weaver Copyright © 2005 – The McGraw-Hill Companies srl Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Gene reporter Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Alcuni promotori eucariotici -140 -120 -100 -80 -60 -40 SV40 (promotore precoce) Timidina kinasi Istone H2B Ottamero CAAT GC TATA -20 1 10 Modulo Consensus TATA box TATAAAA CAAT box GGCCAATCT GC box GGGCGG Ottamero ATTTGCAT " B " ATF " GGGACTTTCC " GTGACGT DNA legato (bp) Fattore >25 22 20 20 23 TFIID CTF/NF1 SP1 Oct-1 Oct-2 60,000 105,000 76,000 52,000 10 10 NFB H2-TF1 44,000 ? ? ? c.linfoidi generale 20 ATF ? ? generale Grandezza Abbond. (dalton) ( /cellula) ? ? 300,000 60,000 ? ? Distrib. generale generale generale generale c.linfoidi ELEMENTI DI RISPOSTA Agente regolatore Modulo Consensus Shock termico Glucocorticoidi Cadmio TPA Siero HSE GRE MRE TRE SRE CNNGCCNNTCCNNG TGGTACAAATGTTCT CGNCCCGGNCNC TGACTCA CCATATTAGG Fattore HSTF Recettore ? AP1 SRF Grandezza (dalton) 93,000 94,000 ? 39,000 52,000 Response elements Response elements may be located in promoters or enhancers. Each response element is recognized by a specific transcription factor. A promoter may have many response elements, which may activate transcription independently or in certain combinations. Biologia molecolare - Robert F. Weaver Copyright © 2005 – The McGraw-Hill Companies srl Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Enhancers Similar sequence elements are found in enhancers and promoters. Enhancers form complexes of transcription factors that interact directly or indirectly with the promoter. Biologia molecolare - Robert F. Weaver Copyright © 2005 – The McGraw-Hill Companies srl Biologia molecolare - Robert F. Weaver Copyright © 2005 – The McGraw-Hill Companies srl Biologia molecolare - Robert F. Weaver Copyright © 2005 – The McGraw-Hill Companies srl Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 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. FATTORI DI TRASCRIZIONE NEGLI EUCARIOTI ( trans-acting factors = fattori che agiscono in trans ) Possono essere distinti in: - Fattori generali - Fattori a monte - Fattori inducibili Assemblaggio del complesso di inizio su un promotore Pol II -5 0 -4 0 -3 0 -2 0 TATA 1 TBP + vari TAF TFII D TATA TFII A TFII B TFIIF Pol II TFII E Pol -1 0 1 10 20 Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005 Le tre RNA polimerasi sono posizionate a livello di tutti i promotori da un fattore che contiene la TBP 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. 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.
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