Meccanismi di Regolazione dell’espressione genica

Fase Nucleare

•Scelta del gene che deve essere espresso

•Maturazione dell’RNA

•Trasferimento Nucleo Citoplasma

 

 

Fase Citoplasmatica

•Sintesi delle catene polipeptidiche

•Modificazioni post-traduzionali

•Trasferimento delle proteine nelle sedi di competenza

Unione di ribonucleotidi monofosfato per formare una catena polinucleotidica

I precursori della sintesi sono i nucleotidi trifosfato.

L’energia che occorre per la formazione del legame fosfodiesterico è data dall’eliminazione del pirofosfato per idrolisi del legame.

ATP ADP

ATP = Adenosin Trifosfato

ADP = Adenosin Difosfato

• ribosomal RNA (rRNA)16S (small ribosomal subunit)23S (large ribosomal subunit)5S (large ribosomal subunit)

• transfer RNA (tRNA)• messenger RNA (mRNA)

Structure of prokaryotic messenger RNA

5’

3’

PuPuPuPuPuPuPuPu AUGShine-Dalgarno sequence initiation

The Shine-Dalgarno (SD) sequence base-pairs with a pyrimidine-rich sequence in 16S rRNA to facilitate the initiation of protein synthesis

Classes of prokaryotic RNA

AAUtermination

translated region

Classes of eukaryotic cellular RNAs

• ribosomal RNA (rRNA)18S (small subunit)28S (large subunit)5.8S (large subunit)5S (large subunit)

• transfer RNA (tRNA)• messenger RNA (mRNA)• heterogeneous nuclear RNA (hnRNA) (precursors of mRNA)• small nuclear RNA (snRNA)

U1, U2, U3, U4, U5, U6, U7, U8, U9, U10...• small cytoplasmic RNA (scRNA)

7SL RNA

What are the enzymes responsible for the synthesis of these RNAs?

The human RNA polymerases

Polymerase Location Product

RNA polymerase I nucleolus 18S, 28S, 5.8S rRNA

RNA polymerase II nucleoplasm hnRNA/mRNA, U1, U2, U4, U5 snRNA

RNA polymerase III nucleoplasm tRNA, 5S RNA, U6 snRNA, 7SL RNA

mitochondrial RNA polymerase mitochondrion all mitochondrial RNA _____________________________________________________________________________________________

Sensitivity of the nuclear RNA polymerases to -amanitin1

RNA pol I resistant RNA pol II high sensitivity (binds with K = 10-8 M) RNA pol III low sensitivity (binds with K = 10-6 M) 1 cyclic octapeptide from the poisonous mushroom Amanita phalloides

Structure of eukaryotic mRNA

7mGpppCap

5’5’ untranslated region

AUGinitiation

translated region

(A)~200

poly(A) tail

3’ untranslated region

UGAtermination

3’AAUAAApolyadenylation signal

• all mRNAs have a 5’ cap and all mRNAs (with the exception of the histone mRNAs) contain a poly(A) tail• the 5’ cap and 3’ poly(A) tail prevent mRNA degradation• loss of the cap and poly(A) tail results in mRNA degradation

5’ 3’

promoter region

exons (filled and unfilled boxed regions)

introns (between exons)

transcribed region

translated region

mRNA structure

+1

b). Gene structure

Legame al promotore della RNA polimerasi

Apertura della doppia elica

Inizio della sintesi

Allungamento

Terminazione

Transcription

RNA polymerase

closed promoter complex

open promoter complex

initiation

elongation

termination

RNA product

Direzione della sintesi

Filamento senso

Filamento antisenso

Sequence elements within a typical eukaryotic gene1

GC TATACAAT GC

-25-50-80-95-130

1 based on the thymidine kinase gene octamertranscription element

promoter

TATA box (TATAAAA)• located approximately 25-30 bp upstream of the +1 start site• determines the exact start site (not in all promoters)• binds the TATA binding protein (TBP) which is a subunit of TFIID

GC box (CCGCCC)• binds Sp1 (Specificity factor 1)

CAAT box (GGCCAATCT)• binds CTF (CAAT box transcription factor)

Octamer (ATTTGCAT)• binds OTF (Octamer transcription factor)

+1

ATTTGCAT

Proteins regulating eukaryotic mRNA synthesis

General transcription factors• TFIID (a multisubunit protein) binds to the TATA box

to begin the assembly of the transcription apparatus• the TATA binding protein (TBP) directly binds the TATA box• TBP associated factors (TAFs) bind to TBP

• TFIIA, TFIIB, TFIIE, TFIIF, TFIIH1, TFIIJ assemble with TFIID

RNA polymerase II binds the promoter region via the TFII’s

Transcription factors binding to other promoter elements and transcription elements interact with proteins at the promoter and further stabilize (or inhibit) formation of a functional preinitiation complex

1TFIIH is also involved in phosphorylation of RNA polymerase II, DNA repair

(Cockayne syndrome mutations), and cell cycle regulation

Nome Alias Chromosoma

TAF1 250 Xq13.1

TAF1L 250like 9p21.1

TAF2 150 8q24.12

TAF3 140 10p15.1

TAF4 135 20q13.33

TAF4B 105 18q11.2

TAF5 100 10q24-10q25.2

TAF6 80 7q22.1

TAF6L 11q12.3

TAF7 55 5q31

TAF7L 50 Xq22.1

TAF8 43 6p21.1

TAF9 32 5q11.2-5q13.1

TAF10 30 11p15.3

TAF11 28 6p21.31

TAF12 20-15 1p35.3

TAF13 18 1p13.3

TAF15 68 17q11.1-q11.2

TF2D TBP + TAF

GTF 2

+1

TBP

TFIID

A

B

E F

H

J

-25

TAFs

Binding of the general transcription factors

• TFIID (a multisubunit protein) binds to the TATA boxto begin the assembly of the transcription apparatus

• the TATA binding protein (TBP) directly binds the TATA box• TBP associated factors (TAFs) bind to TBP

• TFIIA, TFIIB, TFIIE, TFIIF, TFIIH, TFIIJ assemble with TFIID

RNA pol II

TBP

TFIID

A

B

E F

H

J

• RNA polymerase II (a multisubunit protein) binds to the promoter region by interacting with the TFII’s• TFs recruit histone acetylase to the promoter

Binding of RNA polymerase II

Steps in mRNA processing (hnRNA is the precursor of mRNA)• capping (occurs co-transcriptionally)• cleavage and polyadenylation (forms the 3’ end)• splicing (occurs in the nucleus prior to transport)

exon 1 intron 1 exon 2

cap

cap

cap poly(A)

cap poly(A)

Transcription of pre-mRNA and capping at the 5’ end

Cleavage of the 3’ end and polyadenylation

Splicing to remove intron sequences

Transport of mature mRNA to the cytoplasm

Polyadenylation• cleavage of the primary transcript occurs approximately 10-30 nucleotides 3’-ward of the AAUAAA consensus site• polyadenylation catalyzed by poly(A) polymerase• approximately 200 adenylate residues are added

• poly(A) is associated with poly(A) binding protein (PBP)• function of poly(A) tail is to stabilize mRNA

mGpppNmpNmAAUAAA

mGpppNmpNmAAUAAA AA

A

A

AA

3’

cleavage

polyadenylation

Splicing

Rimozione di un introne attraverso due reazioni sequenziali di trasferimento di fosfato, note come transesterificazioni.

Queste uniscono due esoni rimuovendo l’introne come un “cappio”

GENE PREDICTIONGENE PREDICTION

GeneScan GrailEXP Promoter 2.0

Omiga 2.0 GeneMark F GENE SH

Recognition of splice sites• invariant GU and AG dinucleotides at intron ends• donor (upstream) and acceptor (downstream) splice sites

are within conserved consensus sequences

•small nuclear RNA (snRNA) U1 recognizes thedonor splice site sequence (base-pairing interaction)

• U2 snRNA binds to the branch site (base-pairing interaction)

Y= U or C for pyrimidine; N= any nucleotide

G/GUAAGU..................…A.......…YYYYYNYAG/G

donor (5’) splice site acceptor (3’) splice sitebranch site

U1 U2

Chemistry of mRNA splicing• two cleavage-ligation reactions• transesterification reactions - exchange of one

phosphodiester bond for another - not catalyzed bytraditional enzymes• branch site adenosine forms 2’, 5’ phosphodiester bond

with guanosine at 5’ end of intron

G-p-G-U A-G-p-G

2’OH-A

-5’ 3’

intron 1

exon 1 exon 2

Pre-mRNA

First clevage-ligation (transesterification) reaction

branch site adenosine

G-p-G-U A-G-p-G

2’OH-A

-5’ 3’

intron 1

exon 1 exon 2

Step 2: binding of U4, U5, U6

U1

U5

U2U4 U6

G-p-G-U A-G-p-G

2’OH-A

-5’ 3’

intron 1

exon 1 exon 2

Step 3: U1 is released,then U4 is released

U5

U2U6

G-p-G5’ 3’

U-G-5’-p-2’-AA

3’ G-A

intron 1

mRNA

2’OH-A

U5

U2U6

Step 4: U6 binds the 5’ splice site andthe two splicing reactions occur,catalyzed by U2 and U6 snRNPs

G-OH 3’ A-G-p-G

U-G-5’-p-2’-A

5’ 3’A

A

O -

G-p-G5’ 3’

U-G-5’-p-2’-AA

3’ G-A

Splicingintermediate

Lariat

exon 1

exon 1

exon 2

exon 2

intron 1

intron 1

Second clevage-ligation reaction

Spliced mRNA

• ligation of exons releases lariat RNA (intron)

Trans-splicing

Nei protozoi e in un nematode

Differenti molecole di mRNA dallo stesso gene

Splicing alternativo

Uso di promotori alternativi

Uso di segnali di poliadenilazione alternativi

Nei geni degli eucarioti gli enhancers possono distare dalla regione codificante anche più di 50 Kb.

“Enhancers”