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 (carboxy- terminal domain) consisting of multiple repeats of an eptamer.

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

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.

CTCCGAGTCGNNNNNNTGGGCCGCCGG core promoter Upstream Control Element UBF1 SL1 TBP Pol I ? UBF1 SL1 TBP Promotore Pol I del gene per l’RNA ribosomale

Biologia molecolare - Robert F. Weaver Copyright © 2005 – The McGraw-Hill Companies srl

Promotore Pol III interno di tipo 1 Promotore Pol III interno di tipo 2 TFIIIC TFIIIA box A box C TFIIIB Pol III TFIIIB TFIIIC box Abox B Pol III TBP

Gene reporter

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

Alcuni promotori eucariotici OttameroCAATGCTATA SV40 (promotore precoce) Timidina kinasi Istone H2B

Agente regolatore Modulo ConsensusFattore 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 ELEMENTI DI RISPOSTA

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.

Il gene per la metallotioneina (MT) fornisce un esempio di come un gene può essere regolato da circuiti diversi

Un enhancer può contenere vari motivi strutturali riconosciuti da vari fattori

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

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

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.

FATTORI DI TRASCRIZIONE NEGLI EUCARIOTI ( trans-acting factors = fattori che agiscono in trans ) Possono essere distinti in: - Fattori generali - Fattori a monte - Fattori inducibili Fattori specifici

I FATTORI DI TRASCRIZIONE HANNO UN STRUTTURA “MODULARE” COMPOSTA DA DIFFERENTI DOMINI legame al DNA transattivazione dimerizzazione legame al ligando

Biologia molecolare - Robert F. Weaver Copyright © 2005 – The McGraw-Hill Companies srl

modulatoreDNAligando N C hGR hER Recettore “chimera”