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 polymerasesRNA 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

DNA

genepromoter

60/81 bprepeats

spacerpromoter

other repetitiveelements

60/81 bprepeats

spacerpromoter

DNA

RNA

I geni per gli rRNA sono ripetuti in tandem nei genomi eucariotici

rRNA genesrRNA 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

-20 1 20 40-40-60-80-100-120-140-160-180

core promoterUpstream 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 internodi tipo 1

Promotore Pol III internodi tipo 2

TFIIIC

TFIIIA

box A box C

TFIIIB

Pol III

TFIIIB

TFIIICTFIIIC

box A box B

Pol III

TBP

TBP

Gene reporter

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

Alcuni promotori eucariotici

1-20-40-60-80-100 10-120-140

Ottamero CAAT GC TATA

SV40 (promotore precoce)

Timidina kinasi

Istone H2B

Modulo

TATA box

CAAT box

GC box

Ottamero

κB

ATF

TATAAAA

GGCCAATCT

GGGCGG

ATTTGCAT

GGGACTTTCC

GTGACGT

>2522202023101020

TFIID

/ 1CTF NF

1SP

-1Oct

-2Oct

NFκB

2- 1H TF

ATF

60,000105,00076,00052,00044,000

300,00060,000

generale

generale

generale

generale

.c linfoidi

.c linfoidi

generale

generale

ConsensusDNAlegato( )bp

Fattore Grandezza( )dalton

.Abbond( / )cellula

.Distrib

?

??

?

?????

"

"

"

"

Agenteregolatore 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

ELEMENTI DI RISPOSTA

Response elementsResponse 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.

Per vedere questa immagineoccorre QuickTime™ e un

decompressore TIFF.

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

Un enhancer può contenere vari motivi strutturaliriconosciuti da vari fattori

EnhancersEnhancers

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 insulatorsLCR 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

Pol

TATA

-10-20-30-40-50 1 10 20

TATATFII D

TFII A

TFII B

Pol II

TFII E

1 TBP +vari TAF

TFIIF

Assemblaggio delcomplesso di iniziosu un promotorePol 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 promotersInitiation 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 initiationLater 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 HANNOUN STRUTTURA “MODULARE” COMPOSTA DA DIFFERENTI

DOMINI

•legame al DNA

•transattivazi

one

•dimerizzazion

e

•legame al

ligando

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

modulatore DNA ligandoN C

hGR

hER

Recettore “chimera”