RNA

Ribonucleotidi monofosfato uniti a

formare una catena polinucleotidica

Formazione del legame fosfodiesterico

I precursori della sintesi sono i

ribonucleotidi trifosfato.

L’energia che occorre per la formazione del

legame fosfodiesterico

è data dall’eliminazione del pirofosfato per

idrolisi del legame.

La direzione di sintesi è 5’- 3’

La sequenza nucleotidica dell’ RNA è dettata dalla sequenza nucleotidica del DNA

L’ enzima che catalizza l’unione dei

ribonucleotidi è

l’RNA polimerasi

• RNA polimerasi sintetizza RNA in direzione 5’ 3’

• E’ in grado di iniziare la sintesi. Non necessita di un innesco

• Utilizza ribonucleosidi 5’-trifosfato (ATP, GTP, UTP e CTP) e richiede Mg++

• Il 3’OH agisce da nucleofilo sul gruppo fosfato in 5’ del ribonucleoside trifosfato

entrante e si ha liberazione di PPi

• (NMP)n + NTP = (NMP)n+1+ PPi

• Ppi 2Pi

• Ogni nucleotide è selezionato in base alle regole della complementarietà A:U e

G:C

Transcription

RNA polymerase

closed promoter complex

open promoter complex

initiation

elongation

termination

RNA product

Legame al promotore della

RNA polimerasi

Apertura della doppia elica

Inizio della sintesi

Allungamento

Terminazione

Direzione della sintesi

Filamento senso

Filamento antisenso

G C A G T A C A T G T C5' 3'

3' C G T C A T G T A C A G 5' template strand

coding strand

transcription

RNAG C A G U A C A U G U C5' 3'

5’…………..AGAAGATGTCGGGCCAAACGCTCACGGATCGGATCGCCGCCGCTCAGTACAGCGTTACAGGCTCTGCTGT

AGCAAGAGCGGTCTGCAAAGCCACTACTCATGAAGTAATGGGCCCCAAGAAAAAGCACCTGGACTATTTGATCCAGGC

TACCAACGAGACCAATGTTAATATTCCTCAGATGGCCGACACTCTCTTTGAGCGGGCAACAAACAGTAGCTGGGTGGTT

GTGTTTAAGGCTTTAGTGACAACACATCATCTCATGGTGCATGGAAATGAGAGATTTATTCAATATTTGGCTTCTAGAAA

TACACTATTCAATCTCAGCAATTTTTTGGACAAAAGTGGATCCCATGGTTATGATATGTCTACCTTCATAAGGCGCTATA

GTAGATATTTGAATGAAAAGGCTTTTTCTTACAGACAGATGGCCTTTGATTTTGCCAGGGTGAAGAAAGGGGCCGATGG

TGTAATGAGGACAATGGCTCCCGAAAAGCTGCTAAAGAGTATGCCAATACTACAGGGACAAATTGATGCACTGCTTGAA

TTTGATGTGCATCCAAATGAACTAACAAATGGTGTCATAAATGCAGCATTTATGCTTCTTTTCAAAGATCTTATCAAACTT

TTTGCTTGCTACAATGATGGTGTTATTAACTTACTCGAAAAGTTTTTTGAAATGAAGAAAGGACAATGTAAAGATGCTCTA

GAAATTTACAAACGATTTCTAACTAGAATGACACGAGTGTCTGAATTTCTCAAGGTTGCAGAGCAAGTTGGTATTGATAA

AGGTGACATTCCTGACCTCACACAGGCTCCCAGCAGTCTTATGGAGACGCTTGAACAGCATCTAAATACATTAGAAGGA

AAGAAACCTGGAAACAATGAAGGATCTGGTGCTCCCTCTCCATTAAGTAAGTCTTCTCCAGCCACAACTGTTACGTCTC

CTAATTCTACACCAGCTAAAACTATTGACACATCCCCACCGGTTGATTTATTTGCAACTGCATCTGCGGCTGTCCCAGTC

AGCACTTCTAAACCATCTAGTGATCTCCTGGACCTCCAGCCAGACTTTTCCTCTGGAGGGGCAGCAGCAGCCGCAGCA

CCAGCACCACCACCACCTGCTGGAGGAGCCACTGCATGGGGAGACCTTTTGGGAGAGGATTCTTTGGCTGCACTTTCC

TCTGTTCCCTCTGAAGCACAGATTTCAGATCCATTTGCACCAGAACCTACCCCTCCTACTACAACTGCTGAAATTGCAAC

CACTACTGCTGCCACCGCCGCTGCCACCACCACTACCATTCATCTCTTGCCAGCTTAGTAGGCAATCTTGGAATTTCTG

GTACCACAACAAAAAAGGGAGATCTTCAGTGGAATGCTGGAGAGAAAAAGTTGACTGGTGGAGCCAACTGGCAGCCTA

AAGTAGCTCCAGCAACCTGGTCAGCAGGCGTTCCACCAAGTGCACCTTTGCAAGGAGCTGTACCTCCAACCAGTTCAG

TTCCTCCTGTTGCCGGGGCCCCATCGGTTGGACAACCTGGAGCAGGATTTGGAATGCCTCCTGCTGGGACAGGCATG

CCCATGATGCCTCAGCAGCCGGTCATGTTTGCACAGCCCATGATGAGGCCCCCCTTTGGAGCTGCCGCTGTACCTGGC

ACGCAGCTTTCTCCAAGCCCTACACCTGCCAGTCAGAGTCCCAAGAAACCTCCAGCAAAGGACCCATTAGCGGATCTTA

ACATCAAGGATTTCTTGTAAACAATTTAAGCTGCAATATTTGTGACTGAATAGGAAAATAAATGAGTTTGGAGACTTCAAA

TAAGATTGATGCTGAGTTTCAAAGGGAGCCACCAGTACCAAACCCAATACTTACTCATAACTTCTCTTCCAAAATGTGTA

ACACAGCCGTGAAAGTGAACATTAGGAATATGTACTACCTTAGCTGTTATCCCTACTCTTGAAATTGTAGTGTATTTGGA

TTATTTGTGTATTGTACGATGTAAACAATGAATGGATGTTACTGATGCCGTTAGTGCTTTTTTGGACTTCACCTGAGGAC

AGATGATGCAGCTGTTGTGTGGCGAGCTATTTGGAAAGACGTCTGTGTTTTTGAAGGTTTCAATGTACATATAACTTTTG

AACAAACCCCAAACTCTTCCCATAAATTATCTTTTCTTCTGTATCTCTGTTACAAGCGTAGTGTGATAATACCAGATAATA

AGGAAAACACTCATAAATATACAAAACTTTTTCAGTGTGGAGTACATTTTTCCAATCACAGGAACTTCAACTGTTGTGAGA

AATGTTTATTTTTGTGGCACTGTATATGTTAA…..3’

core enzymeholoenzyme

Holoenzyme

The holoenzyme of RNA-pol in E.coli

consists of 5 different subunits: 2 .

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

5’ 3’

promoter

region

exons (filled and unfilled boxed regions)

introns (between exons)

transcribed region

translated region

mRNA structure

+1

b). Gene structure

Sequence elements within a typical eukaryotic gene1

GC TATACAAT GC

-25-50-80-95-130

1 based on the thymidine kinase gene

octamer

transcription

elementpromoter

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

+1

TBP

TFIID

A

B

EF

H

J

-25

TAFs

Binding of the 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, TFIIH, TFIIJ assemble with TFIID

RNA pol II

TBP

TFIID

A

B

EF

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

TATA BOX

TBP

Saddle-like

domain

TATA BOX BINDING PROTEIN

DNA

BINDING

TAF3

TAF10

TAF6 TAF11

TAF12

TAF4

TATA BOX

TBP

TAF5

TAF10

TAF8

TAF5

TAF4

TAF12TAF13TAF9 TA

F3

TAF11

TAF6TAF13 TAF9

TAF8

TAF7

TAF1: Acetyl

transferase activity

Interaction with

TFIIF

TAF5 stabilizes

TAFs interaction,

specially histone-

like ones (TAF6,

TAF9)

DNA BENDING

TFIID

Pre-initiation complex (PIC)

RNA pol II

TF II F

TBP TAF

TATADNA

TF II

ATF II

B

TF II E

TF II H

• TBP of TFII D binds TATA

• TFII A and TFII B bind TFII D

• TFII F-RNA-pol complex binds TFII B

• TFII F and TFII E open the dsDNA

(helicase and ATPase)

• TFII H: completion of PIC

Pre-initiation complex (PIC)

• The termination sequence is AATAAA

followed by GT repeats.

• The termination is closely related to

the post-transcriptional modification.

c. Termination

Structure of eukaryotic mRNA

7mGppp

Cap

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

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

• The 5- cap structure is found on

hnRNA too. The capping process

occurs in nuclei.

• The cap structure of mRNA will be

recognized by the cap-binding protein

required for translation.

• The capping occurs prior to the

splicing.

b. Poly-A tailing at 3 - end

• There is no poly(dT) sequence on the

DNA template. The tailing process

dose not depend on the template.

• The tailing process occurs prior to the

splicing.

• The tailing process takes place in the

nuclei.

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

mGpppNmpNm

AAUAAA

mGpppNmpNm

AAUAAA 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”

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 the

donor 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

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

U2

U4 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 and

the two splicing reactions occur,

catalyzed by U2 and U6 snRNPs

Differenti molecole di mRNA dallo stesso gene

Splicing alternativo

Uso di promotori alternativi

Uso di segnali di poliadenilazione alternativi

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

AAUtermination

translated region

Il gene dei procarioti è policistronico

Nei geni degli eucarioti gli enhancers possono distare dalla regione

codificante anche più di 50 Kb.

“Enhancers”

Punto 1

Organizzazione della cromatina

Inizio della trascrizione

Regolazione dell’espressione genica

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

Il differenziamento cellulare dipende

da meccanismi di regolazione

dell’espressione genica

“Trascrizione”

sintesi di tutti gli RNA cellulari