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? 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 _______________________________________________________________ ______________________________ 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. • Legame al promotore della RNA polimerasi • Apertura della doppia elica • Inizio della sintesi • Allungamento • Terminazione Direzione della sintesi Filamento senso Filamento antisenso closed promoter complex RNA polymerase open promoter complex initiation elongation termination RNA product Transcription 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 Binding of the general transcription factors E F TAFs TFIID B H TBP A J +1 -25 • 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 Binding of RNA polymerase II E F 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 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 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” b). Gene structure promoter region exons (filled and unfilled boxed regions) +1 introns (between exons) transcribed region mRNA structure 5’ 3’ translated region Structure of eukaryotic mRNA Cap 5’ 7mGppp initiation 5’ untranslated region AUG 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 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 cleavage AAUAAA mGpppNmpNm AAUAAA mGpppNmpNm A A A polyadenylation A A A 3’ • poly(A) is associated with poly(A) binding protein (PBP) • function of poly(A) tail is to stabilize mRNA “Enhancers” Nei geni degli eucarioti gli enhancers possono distare dalla regione codificante anche più di 50 Kb.
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