Meccanismi di Regolazione dell’espressione genica

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

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

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 PuPuPuPuPuPuPuPu AUG 5’ translated region AAU 3’ 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?

9. The human RNA polymerases PolymeraseLocationProduct 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 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

14. b). Gene structure promoter region exons (filled and unfilled boxed regions) +1 introns (between exons) transcribed region mRNA structure 5’ 3’ translated region

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

16. Transcription closed promoter complex RNA polymerase open promoter complex initiation elongation termination RNA product

17. Direzione della sintesi Filamento senso Filamento antisenso

19. Sequence elements within a typical eukaryotic gene1 1based on the thymidine kinase gene octamer transcription element +1 promoter GC CAAT GC TATA ATTTGCAT -130 -95 -80 -50 -25 • 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)

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

21. TF2D TBP + TAF

22. GTF 2

24. Binding of the general transcription factors F TAFs E TFIID B H TBP A J -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 F E TFIID B H TBP A J 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

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 1 intron 1 exon 2 Transcription of pre-mRNA and capping at the 5’ end cap Cleavage of the 3’ end and polyadenylation cap cap poly(A) Splicing to remove intron sequences cap poly(A) Transport of mature mRNA to the cytoplasm

28. Polyadenylation • cleavage of the primary transcript occurs approximately • 10-30 nucleotides 3’-ward of the AAUAAA consensus site • polyadenylation catalyzed bypoly(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 polyadenylation A AAUAAA A A A mGpppNmpNm A A 3’

30. 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”

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

38. 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

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 branch site adenosine 2’OH-A exon 1 exon 2 5’ G-p-G-U A-G-p-G - 3’ First clevage-ligation (transesterification) reaction

40. U2 U2 U5 intron 1 Step 2: binding of U4, U5, U6 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 2’OH-A U6 exon 1 exon 2 5’ G-p-G-U A-G-p-G - 3’

41. 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 U-G-5’-p-2’-A A 3’ G-A mRNA 5’ G-p-G 3’

42. ligation of exons releases lariat RNA (intron) intron 1 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 U-G-5’-p-2’-A A 3’ G-A Spliced mRNA exon 1 exon 2 5’ G-p-G 3’

44. Trans-splicing Nei protozoi e in un nematode

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