2 Meccanismi di Regolazione dell’espressione genica Fase NucleareScelta del gene che deve essere espressoMaturazione dell’RNATrasferimento Nucleo CitoplasmaFase CitoplasmaticaSintesi delle catene polipeptidicheModificazioni post-traduzionaliTrasferimento delle proteine nelle sedi di competenza
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 ADPATP = Adenosin TrifosfatoADP = Adenosin Difosfato
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 RNAShine-Dalgarno sequenceinitiation5’PuPuPuPuPuPuPuPuAUGThis 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 ribosomalRNA. 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 region3’AAUterminationThe Shine-Dalgarno (SD) sequence base-pairs with a pyrimidine-richsequence 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 RNAWhat 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 areprecursors of mRNAs. They are of heterogeneous size because they represent RNAs in various stages ofprocessing. There are numerous species of small nuclear and small cytoplasmic RNAs. We will be examiningthe functions of U1, U2, U4, U5, and U6 snRNAs in mRNA processing, and 7SL scRNA in the synthesis ofmembrane-bound and secreted proteins.
9 The human RNA polymerases Polymerase Location ProductRNA polymerase I nucleolus S, 28S, 5.8S rRNARNA polymerase II nucleoplasm hnRNA/mRNA,U1, U2, U4, U5 snRNARNA polymerase III nucleoplasm tRNA, 5S RNA,U6 snRNA, 7SL RNAmitochondrialRNA polymerase mitochondrion all mitochondrial RNA_____________________________________________________________________________________________Sensitivity of the nuclear RNA polymerases to a-amanitin1RNA pol I resistantRNA 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 Capinitiation5’ untranslated region5’AUG7mGppptranslated regionUGAtermination3’ untranslated regionpolyadenylation signalAAUAAA(A)~2003’poly(A) tailall mRNAs have a 5’ cap and all mRNAs (with the exceptionof the histone mRNAs) contain a poly(A) tailthe 5’ cap and 3’ poly(A) tail prevent mRNA degradationloss of the cap and poly(A) tail results in mRNA degradation
14 b). Gene structure promoter region exons (filled and unfilled boxed regions)+1introns (between exons)transcribed regionThis slide shows the structure of a typical human gene and its corresponding messenger RNA (mRNA). Mostgenes 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 transcribedregion of a gene (double-ended arrow) starts at the +1 nucleotide at the 5' end of the first exon and includes allof the exons and introns (initiation of transcription is regulated by the promoter region of a gene, which isupstream 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 (theregion 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 structure5’3’translated region
15 Legame al promotore della RNA polimerasi Apertura della doppia elicaInizio della sintesiAllungamentoTerminazione
16 Transcription RNA polymerase closed promoter complex open promoter complexinitiationelongationThis figure shows an overview of the main steps in transcription. RNA polymerase binds the promoter firstforming 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 templatestrand is transcribed into a complementary nucleotide in the growing polynucleotide chain. Termination occursat the end of the gene. At this point the RNA product is complete and the complex dissociates.terminationRNA product
17 Direzione della sintesi Filamento sensoFilamento antisenso
19 Sequence elements within a typical eukaryotic gene1 1 based on the thymidine kinase geneoctamertranscriptionelement+1promoterATTTGCATGCCAATGCTATA-130-95-80-50-25TATA box (TATAAAA)located approximately bp upstream of the +1 start sitedetermines the exact start site (not in all promoters)binds the TATA binding protein (TBP) which is a subunit of TFIIDGC 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. colipromoter). There is a TATA box located approximately bp upstream of the start site. It interacts with theTATA binding protein and determines the exact site of initiation. A little further upstream are two GC boxes (ininverted orientation with respect to each other) and a CAAT box. These promoter elements are all consensussequences. Still further upstream is an octomer element. Transcription factors specific to each of theseelements function by recognizing these DNA sequences and binding to these DNA sites.
20 Proteins regulating eukaryotic mRNA synthesis General transcription factorsTFIID (a multisubunit protein) binds to the TATA boxto begin the assembly of the transcription apparatusthe TATA binding protein (TBP) directly binds the TATA boxTBP associated factors (TAFs) bind to TBPTFIIA, TFIIB, TFIIE, TFIIF, TFIIH1, TFIIJ assemble with TFIIDRNA polymerase II binds the promoter region via the TFII’sTranscription factors binding to other promoter elements andtranscription elements interact with proteins at the promoterand further stabilize (or inhibit) formation of a functionalpreinitiation complex1TFIIH is also involved in phosphorylation of RNA polymerase II, DNA repair(Cockayne syndrome mutations), and cell cycle regulationProteins that regulate transcription are called transcription factors. The general transcription factors functionat all mRNA promoters and are abbreviated TFII for transcription factors regulating RNA polymerase IIpromoters. Each (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIJ) is a multisubunit protein. They function torecognize and bind to the promoter so as to help stabilize the binding of RNA polymerase. They also bind toother transcription factors through protein-protein interactions, which are bound to their own specific DNAelements.
21 TF2D TBP + TAF Nome Alias Chromosoma TAF1 250 Xq13.1 TAF1L 250like 1508q24.12TAF314010p15.1TAF413520q13.33TAF4B10518q11.2TAF510010q24-10q25.2TAF6807q22.1TAF6L11q12.3TAF7555q31TAF7L50Xq22.1TAF8436p21.1TAF9325q11.2-5q13.1TAF103011p15.3TAF11286p21.31TAF1220-151p35.3TAF13181p13.3TAF156817q11.1-q11.2TF2D TBP + TAF
24 Binding of the general transcription factors TAFsETFIIDBHTBPAJThe next series of figures shows the sequential building of the preinitiation complex. It starts with thebinding of TBP to the TATA box.-25+1TFIID (a multisubunit protein) binds to the TATA boxto begin the assembly of the transcription apparatusthe TATA binding protein (TBP) directly binds the TATA boxTBP associated factors (TAFs) bind to TBPTFIIA, TFIIB, TFIIE, TFIIF, TFIIH, TFIIJ assemble with TFIID
25 Binding of RNA polymerase II TFIIDBHTBPAJThe binding of the general transcription factors then promotes binding of RNA polymerase II. The TF's alsorecruit histone acetylase to the promoter to help destablize (relax) nucleosomes, which are present on the DNA(but not shown).RNA pol IIRNA polymerase II (a multisubunit protein) binds tothe promoter region by interacting with the TFII’sTFs recruit histone acetylase to the promoter
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 2Transcription of pre-mRNA and capping at the 5’ endcapCleavage of the 3’ end and polyadenylationcapThis figure provides an overview of the major steps in eukaryotic mRNA processing. The mechanisms will beexamined in more detail in the first part of this lecture.cappoly(A)Splicing to remove intron sequencescappoly(A)Transport of mature mRNA to the cytoplasm
28 cleavage of the primary transcript occurs approximately Polyadenylationcleavage of the primary transcript occurs approximately10-30 nucleotides 3’-ward of the AAUAAA consensus sitepolyadenylation catalyzed by poly(A) polymeraseapproximately 200 adenylate residues are addedpoly(A) is associated with poly(A) binding protein (PBP)function of poly(A) tail is to stabilize mRNAcleavageAAUAAAmGpppNmpNmThe 3' end of mRNA is not determined by termination of transcription. Transcription continues downstream tosome not well-defined termination point. Shortly after the AAUAAA polyadenylation signal has beentranscribed, a nuclease cleaves the transcript at a site approximately nucletides downstream of theAAUAAA signal. Poly(A) polymerase then starts the synthesis of a poly(A) sequence by the stepwise additionof 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 nucleotidesubstitution in the 3' UTR, which is present in 1-2% of the population. This mutation increases the efficiency of3' end processing, leading to excess production of thrombin mRNA and protein (Mendell and Dietz, Cell107:411; 2001). Pulmonary embolism is the most common cause, in the industrialized world, of maternal deathduring pregnancy or in the period following delivery. About 70% of women who present with venousthromboembolism during pregnancy are carriers of hereditary or acquired thrombophilia (Eldor, J ThrombThrombolysis 12:23; 2001).AAUAAAApolyadenylationAAmGpppNmpNmAAA3’
30 Queste uniscono due esoni rimuovendo l’introne come un “cappio” SplicingRimozione di un introne attraverso due reazioni sequenziali di trasferimento di fosfato, note come transesterificazioni.Queste uniscono due esoni rimuovendo l’introne come un “cappio”
38 U1 U2 Recognition of splice sites invariant GU and AG dinucleotides at intron endsdonor (upstream) and acceptor (downstream) splice sitesare within conserved consensus sequencessmall 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 nucleotidedonor (5’) splice sitebranch siteacceptor (3’) splice siteG/GUAAGU …A …YYYYYNYAG/GU1U2While the splicing reaction per se is carried out by RNA chemistry, there are a number of exogenous proteinand protein-RNA factors that need to be involved in the reaction. These are involved in the recognition of thesplice 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 areG/GUAAGU and YYYYYYYYYYNYAG/G, where Y is a pyrimidine and N is any nucleotide. The very ends of theintron are flanked by invariant GU and AG dinucleotides, with the rest of the consensus sequence in each casebeing a close match to the consensus but having some variation. The small nuclear RNA U1recognizes the donor splice site through a base pairing interaction with the consensus sequence. The smallnuclear 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 onephosphodiester bond for another - not catalyzed bytraditional enzymesbranch site adenosine forms 2’, 5’ phosphodiester bondwith guanosine at 5’ end of intronintron 1Pre-mRNARemoval of an intron transcript is carried out by mRNA splicing. This is accomplished by twocleavage-ligation reactions: one that cleaves at the 5' end of the intron and the second that cleaves at the 3' endof the intron. Each of these reactions is a so-called transesterification reaction, that is, one phosphodiesterbond is exchanged for another. Thus, because bond energy is preserved, there is no need for additional inputenergy for these reactions. Furthermore, these reactions are not catalyzed by protein enzymes - they aremediated 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 partof the RNA polynucleotide chain, its 3' hydroxyl is already involved in a covalent bond (the 3', 5' phosphodiesterbond). 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. Thestructure is shown in the next figure.2’OH-Abranch site adenosineexon 1exon 25’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 1Step 2: binding of U4, U5, U6U22’OH-AU4 U6exon 1exon 2U55’G-p-G-U A-G-p-G-3’U1Step 3: U1 is released,then U4 is releasedintron 1Step 2 involves binding of U4, U5, and U6 snRNPs. Rearrangement of the spliceosome then takes place instep 3, U1 and U4 are released, and U6 shifts to the donor splice site.U22’OH-AU6exon 1exon 2U55’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 snRNPsintron 12’OH-AU2U6U-G-5’-p-2’-AAU5mRNA3’ G-AIn step 4, the reacting groups are brought into close proximity and the two transesterification reactions takeplace, catalyzed by U2 and U6 snRNPs. U5 serves a bridging function by interacting with the 5' exon and withthe 3' exon immediately adjacent to the splice sites.5’G-p-G3’
42 ligation of exons releases lariat RNA (intron) SplicingintermediateU-G-5’-p-2’-AAexon 1exon 25’G-OH 3’ A-G-p-GOA-3’Second clevage-ligation reactionintron 1LariatThe 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 secondclevage-ligation reaction occurs by attack by the 3' hydoxyl at the 3' end of the upstream exon on the 3' end ofthe intron. This simultaneously releases the intron transcript (the so-called lariat) and ligates the two exonstogether.U-G-5’-p-2’-AA3’ G-ASpliced mRNAexon 1exon 25’G-p-G3’