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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
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