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Meccanismi di Regolazione dell’espressione genica

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2 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

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4 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.

5 ATP ADP ATP = Adenosin Trifosfato ADP = Adenosin Difosfato

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7 Classes of prokaryotic RNA
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 Shine-Dalgarno sequence initiation 5’ PuPuPuPuPuPuPuPu AUG This figure reviews the major classes of prokaryotic RNA. Note that prokaryotic mRNAs have a purine-rich (Pu) Shine-Dalgarno sequence to facilitate the initiation of translation by base pairing with the 16S ribosomal RNA. This sequence is not present in eukaryotic mRNAs. The translated region of the mRNA is shown in red. The presence of the Shine-Dalgarno sequence makes possible internal initiation in polycistronic mRNAs. translated region 3’ AAU termination The Shine-Dalgarno (SD) sequence base-pairs with a pyrimidine-rich sequence in 16S rRNA to facilitate the initiation of protein synthesis

8 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? In addition to the three major classes of RNA (rRNA, tRNA, and mRNA), which are also present in prokaryotes, eukaryotes have several other RNAs. Heterogeneous nuclear RNA is the population of nuclear RNAs that are precursors of mRNAs. They are of heterogeneous size because they represent RNAs in various stages of processing. There are numerous species of small nuclear and small cytoplasmic RNAs. We will be examining the functions of U1, U2, U4, U5, and U6 snRNAs in mRNA processing, and 7SL scRNA in the synthesis of membrane-bound and secreted proteins.

9 The human RNA polymerases
Polymerase Location Product RNA polymerase I nucleolus S, 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 a-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

10 Structure of eukaryotic mRNA
Cap initiation 5’ untranslated region 5’ AUG 7mGppp translated region UGA termination 3’ untranslated region polyadenylation signal AAUAAA (A)~200 3’ poly(A) tail 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

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14 b). Gene structure promoter region
exons (filled and unfilled boxed regions) +1 introns (between exons) transcribed region This slide shows the structure of a typical human gene and its corresponding messenger RNA (mRNA). Most genes in the human genome are called "split genes" because they are composed of "exons" separated by "introns." The exons are the regions of genes that encode information that ends up in mRNA. The transcribed region of a gene (double-ended arrow) starts at the +1 nucleotide at the 5' end of the first exon and includes all of the exons and introns (initiation of transcription is regulated by the promoter region of a gene, which is upstream of the +1 site). RNA processing (the subject of a another lecture) then removes the intron sequences, "splicing" together the exon sequences to produce the mature mRNA. The translated region of the mRNA (the region that encodes the protein) is indicated in blue. Note that there are untranslated regions at the 5' and 3' ends of mRNAs that are encoded by exon sequence but are not directly translated. mRNA structure 5’ 3’ translated region

15 Legame al promotore della RNA polimerasi
Apertura della doppia elica Inizio della sintesi Allungamento Terminazione

16 Transcription RNA polymerase closed promoter complex
open promoter complex initiation elongation This figure shows an overview of the main steps in transcription. RNA polymerase binds the promoter first forming a closed promoter complex and then forming an open promoter complex with the localized melting (strand separation) of the DNA. Initiation then occurs by the synthesis of the first phosphodiester bond. Elongation proceeds by translocation of RNA polymerase down the gene. Each nucleotide of the template strand is transcribed into a complementary nucleotide in the growing polynucleotide chain. Termination occurs at the end of the gene. At this point the RNA product is complete and the complex dissociates. termination RNA product

17 Direzione della sintesi
Filamento senso Filamento antisenso

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19 Sequence elements within a typical eukaryotic gene1
1 based on the thymidine kinase gene octamer transcription element +1 promoter ATTTGCAT GC CAAT GC TATA -130 -95 -80 -50 -25 TATA box (TATAAAA) located approximately 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) This figure shows the regulatory elements for a "typical" eukaryotic gene (compare this to the E. coli promoter). There is a TATA box located approximately bp upstream of the start site. It interacts with the TATA binding protein and determines the exact site of initiation. A little further upstream are two GC boxes (in inverted orientation with respect to each other) and a CAAT box. These promoter elements are all consensus sequences. Still further upstream is an octomer element. Transcription factors specific to each of these elements function by recognizing these DNA sequences and binding to these DNA sites.

20 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 Proteins that regulate transcription are called transcription factors. The general transcription factors function at all mRNA promoters and are abbreviated TFII for transcription factors regulating RNA polymerase II promoters. Each (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIJ) is a multisubunit protein. They function to recognize and bind to the promoter so as to help stabilize the binding of RNA polymerase. They also bind to other transcription factors through protein-protein interactions, which are bound to their own specific DNA elements.

21 TF2D TBP + TAF Nome Alias Chromosoma TAF1 250 Xq13.1 TAF1L 250like
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

22 GTF 2

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24 Binding of the general transcription factors
TAFs E TFIID B H TBP A J The next series of figures shows the sequential building of the preinitiation complex. It starts with the binding of TBP to the TATA box. -25 +1 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

25 Binding of RNA polymerase II
TFIID B H TBP A J The binding of the general transcription factors then promotes binding of RNA polymerase II. The TF's also recruit histone acetylase to the promoter to help destablize (relax) nucleosomes, which are present on the DNA (but not shown). RNA pol II RNA polymerase II (a multisubunit protein) binds to the promoter region by interacting with the TFII’s TFs recruit histone acetylase to the promoter

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27 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 intron exon 2 Transcription of pre-mRNA and capping at the 5’ end cap Cleavage of the 3’ end and polyadenylation cap This figure provides an overview of the major steps in eukaryotic mRNA processing. The mechanisms will be examined in more detail in the first part of this lecture. cap poly(A) Splicing to remove intron sequences cap poly(A) Transport of mature mRNA to the cytoplasm

28 cleavage of the primary transcript occurs approximately
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 cleavage AAUAAA mGpppNmpNm The 3' end of mRNA is not determined by termination of transcription. Transcription continues downstream to some not well-defined termination point. Shortly after the AAUAAA polyadenylation signal has been transcribed, a nuclease cleaves the transcript at a site approximately nucletides downstream of the AAUAAA signal. Poly(A) polymerase then starts the synthesis of a poly(A) sequence by the stepwise addition of adenylate residues producing a poly(A) tail of approximately 200 residues. Hereditary thrombophilia (increased tendency for the blood to clot) is caused by a single nucleotide substitution in the 3' UTR, which is present in 1-2% of the population. This mutation increases the efficiency of 3' end processing, leading to excess production of thrombin mRNA and protein (Mendell and Dietz, Cell 107:411; 2001). Pulmonary embolism is the most common cause, in the industrialized world, of maternal death during pregnancy or in the period following delivery. About 70% of women who present with venous thromboembolism during pregnancy are carriers of hereditary or acquired thrombophilia (Eldor, J Thromb Thrombolysis 12:23; 2001). AAUAAA A polyadenylation A A mGpppNmpNm A A A 3’

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

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34 GENE PREDICTION GeneScan GrailEXP Promoter 2.0 Omiga 2.0 GeneMark
F GENE SH

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38 U1 U2 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 donor (5’) splice site branch site acceptor (3’) splice site G/GUAAGU …A …YYYYYNYAG/G U1 U2 While the splicing reaction per se is carried out by RNA chemistry, there are a number of exogenous protein and protein-RNA factors that need to be involved in the reaction. These are involved in the recognition of the splice sites and in helping to fold the intron into a proper configuration for the splicing reaction. The upstream (donor) and downstream (acceptor) splice sites are made up of consensus sequences that are G/GUAAGU and YYYYYYYYYYNYAG/G, where Y is a pyrimidine and N is any nucleotide. The very ends of the intron are flanked by invariant GU and AG dinucleotides, with the rest of the consensus sequence in each case being a close match to the consensus but having some variation. The small nuclear RNA U1 recognizes the donor splice site through a base pairing interaction with the consensus sequence. The small nuclear RNA U2 recognizes the branch site, also through a base pairing interaction.

39 Chemistry of mRNA splicing two cleavage-ligation reactions
transesterification reactions - exchange of one phosphodiester bond for another - not catalyzed by traditional enzymes branch site adenosine forms 2’, 5’ phosphodiester bond with guanosine at 5’ end of intron intron 1 Pre-mRNA Removal of an intron transcript is carried out by mRNA splicing. This is accomplished by two cleavage-ligation reactions: one that cleaves at the 5' end of the intron and the second that cleaves at the 3' end of the intron. Each of these reactions is a so-called transesterification reaction, that is, one phosphodiester bond is exchanged for another. Thus, because bond energy is preserved, there is no need for additional input energy for these reactions. Furthermore, these reactions are not catalyzed by protein enzymes - they are mediated by the reacting RNAs themselves. The first of these two phosphotransfer reactions is shown here. This involves the branch site adenosine, which attacks the 3', 5' phosphodiester bond at the 5' end of the intron. Since the branch site adenosine is part of the RNA polynucleotide chain, its 3' hydroxyl is already involved in a covalent bond (the 3', 5' phosphodiester bond). Therefore, it utilizes its 2' hydroxyl for this reaction. Attack by this 2' hydroxyl breaks the bond at the 5' end of the intron by forming a bond between the 5' end of the intron and this branch site adenosine. The structure is shown in the next figure. 2’OH-A branch site adenosine exon 1 exon 2 5’ G-p-G-U A-G-p-G - 3’ First clevage-ligation (transesterification) reaction

40 U2 U4 U6 U5 U1 U2 U6 U5 Step 2: binding of U4, U5, U6
intron 1 Step 2: binding of U4, U5, U6 U2 2’OH-A U4 U6 exon 1 exon 2 U5 5’ G-p-G-U A-G-p-G - 3’ U1 Step 3: U1 is released, then U4 is released intron 1 Step 2 involves binding of U4, U5, and U6 snRNPs. Rearrangement of the spliceosome then takes place in step 3, U1 and U4 are released, and U6 shifts to the donor splice site. U2 2’OH-A U6 exon 1 exon 2 U5 5’ G-p-G-U A-G-p-G - 3’

41 U2 U6 U5 Step 4: U6 binds the 5’ splice site and
the two splicing reactions occur, catalyzed by U2 and U6 snRNPs intron 1 2’OH-A U2 U6 U-G-5’-p-2’-A A U5 mRNA 3’ G-A In step 4, the reacting groups are brought into close proximity and the two transesterification reactions take place, catalyzed by U2 and U6 snRNPs. U5 serves a bridging function by interacting with the 5' exon and with the 3' exon immediately adjacent to the splice sites. 5’ G-p-G 3’

42 ligation of exons releases lariat RNA (intron)
Splicing intermediate U-G-5’-p-2’-A A exon 1 exon 2 5’ G-OH 3’ A-G-p-G O A - 3’ Second clevage-ligation reaction intron 1 Lariat The intermediate produced by the first transesterification reaction involves a branching polynucleotide (hence, branch site adenosine) where the adenosine makes bonds with both its 2' OH and its 3' OH. The second clevage-ligation reaction occurs by attack by the 3' hydoxyl at the 3' end of the upstream exon on the 3' end of the intron. This simultaneously releases the intron transcript (the so-called lariat) and ligates the two exons together. U-G-5’-p-2’-A A 3’ G-A Spliced mRNA exon 1 exon 2 5’ G-p-G 3’

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44 Nei protozoi e in un nematode
Trans-splicing Nei protozoi e in un nematode

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50 Differenti molecole di mRNA dallo stesso gene
Splicing alternativo Uso di promotori alternativi Uso di segnali di poliadenilazione alternativi

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

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