11 주 RNA 합성 11.1 DNA-dependent synthesis of RNA: Bacteria에서의 transcription l RNA polymerase (5 subunits로구성 : 2α, β, β, σ) l 효소에의한 RNA 합성의특징 - Complementary sequence to template DNA - RNA chain의합성방향 : 5 3 - 두 strands의일시적분리를통하여, transcription bubble을형성 - RNA polyemrase는 primer없이 transcription을시작함. 맨앞 (5 ) 의 nucletotide는 triphosphate를가짐. - Ribonucletoside-5 -triphosphate (rntp) - ATP, GTP, CTP, UTP를 precursor 로사용 - RNA polymerase는 3 5 exonuclease activity를가지지않음. l RNA polymerase의구성 : 5 subunits로구성 - Core enzyme: 2α, β, β - σ: Transcription initiation 후분리됨. l Transcription signals 1 Promoters: RNA polyemrase의결합위치 Transcription initiation site positioning: core sequence를가짐. (-10 sequence, -35 sequence) - Consensus sequence a) -10 region: (5 ) TATAAT (3 ) b) -35 region: (5 ) TTGACA (3 ) RNA polymerase의 σ 70 subunit가이 consensus sequence와 interaction - Promoter sequence의 영향 : 유전자 발현의 조절 효과 (Transcription regulation)
l Steps in the initiation of transcription - RNA polymerase + promoter - Promoter-polymerase complex (Closed complex) 형성 - Promoter-polymerase complex (Open complex) 형성 - Promoter clearance (σ factor의분리 ) 2 Termination l Intrinsic terminator - Transcription을 terminate하는특정 sequence Inverted-repeat sequence 를가짐예 ) ABCDEF XYZ F E D C B A - Complex Isomerization: conformational change New RNA transcript는 hairpin 구조형성 RNA-DNA hybrid, RNA-polymerase interaciton 약화 RNA의 dissociation (termination) l Rho (ρ)-dependent terminator - ρ protein이 RNa의 specific binding site에결합하여 5 3 방향으로이동. - Transcription complex (paused at a termination sites) 를만나면, RNA transcript를 release하도록함. - ρ protein은 ATP-dependent RNA-DNA helicase activity를가짐. 11.2 DNA-dependent synthesis of RNA: 진핵세포에서의 transcription l Three kinds of RNA polymerases - Class I: 18S, 5.8S, 28S rrnas - Class II: mrna - Clasee III: trna, 5S rrna l 일반적인 eukaryotic promoter 의구조 - TATA box: -30 region 에존재, transcription 출발위치및 tnrascription 속도
를결정 - Initiator: +1 전후에존재. Transcription 출발위치결정 - 다양한 upstream regulatory sequences: upstream 쪽에는매우다양한 cis-regulatory element들이존재하여, 다양한 transcription factor에의한유전자발현의조절이이루어짐. l RNA polymerase II에의한 transcription에필요한 protein들 - General transcription factors: TFIID, TFIIB, TFIIF, TFIIE, TFIIH, TFIIA - 위의 general transcription factors들은일정한순서로 promoter에결합함 : TFIID TFIIA TFIIF + PolII TFIIE TFIIH l Promoter - Transcription factors/rna polymerase들이결합하여복합체를형성함 - Initiation 과정중 RNA polyemrase II의 C-terminal domain에과인산화 (hyperphosphorylation) 가이루어짐. - 조절 : 다양한 transcription factor 들에의해조절됨. 11.3 RNA processing l Splicing - Intron은 primary transcript의 50-90% 를차지함. - Splicing은 transcription과 capping 후일어남. - Polyadenylation 전또는후에 splicing이일어남. 1) Capping l 5 -end에 transcription initiation 직후에일어남 l 7-Methylguanosine (7-MeG): Primary transcript의 5 -terminal nucleotide에 5-5 linkage로연결됨. l Capping의효과 - Nucleases에의한 degradation으로부터 RNA를보호 - Protein-synthesizing machienry가인지할수있는위치제공 ribosome의결합을용이하게함.
2) Splicing l Four classes of introns - Group I: Self-splicing - Group II: Self-splicing - snrnp mediated - Endonuclease mediated l Group I splicing - 일부핵내유전자, 미토콘드리아, 엽록체내유전자들에서전사된 RNA에서일어남. - Guanine nucleoside or nucleotide cofactor가필요 - Ribose 3 -hydroxyl group이 phosphorus에 nucleophilic attack 새로운 phosphodiester bond 형성 Two transesterification reaction으로 splicing 진행 l Group II splicing - 미코톤드리아, 엽록체 mrna에서주로일어남. - Nucleophile이 guanine nucleotide가아닌 intron 내의 adenylate residue의 2 -OH group임. - Ribose 2 -hydroxyl group이 phosphorus에 nucleophilic attack 새로운 phosphodiester bond 형성 Two transesterification reaction으로 splicing 진행 l Small nuclear ribonucleoprotein (snrnp) mediated splicing - 핵내 mrna primary transcripts에서주로일어남. - snrnp: small nuclear RNA (snrna) 를포함. U1 5 splice site 주변 sequence에 complementary sequence U2 nucleophile로작용할 adnylate를포함한 sequence에결합. - Splicesome: snrnp 간의 interaction으로 splicing site에형성됨 (5 RNA + 50 proteins) l Endonuclease mediated splicing: in certain trnas
3) Polyadenylation l Poly(A) tail의효과 - mrna의 stability 증가 - mrna의 translation 속도의증가 l Differential RNA processing - 하나의유전자로부터여러종류의단백질의생산이가능함. - Alternative cleavage and polyadenylation - Alternative splicing