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1 제 11 장. 핵산의생합성 : 전사 (DNA transcription)

2 Transcription and Translation ( 진핵생물 ) ( 원핵생물 ) RNA processing 없음 RNA processing 있음

3 Messenger RNA (mrna): 아미노산순서를결정한다. Alanine Serine Glycine Isoleucine GCA UCC GGC AUC 각아미노산은 mrna 의 codon 에의해정해진다. Codon 은 mrna 에서세글자의 code 이다.

4 Transfer RNA (trna) Alanine C G U Anticodon 리보솜에서 mrna 의암호 (codon) 에따라아미노산을 mrna 에운반한다. Anticodon 은 codon 과상보적이다.

5 단백질합성 : 리보소옴 리보소옴 trna Alanine Serine CGU AGG GCA UCC GGC AUC mrna

6 단백질합성 : 리보소옴 리보소옴 trna Alanine Serine Glycine Isoleucin e CGU AGG CCG UAG GCA UCC GGC AUC mrna

7 전사 : E. Coli RNA Polymerase Template strand = antisense Coding strand = sense Figure 10.1 The basics of replication

8 전사촉진유전자 (Promoter) sequence +1 : 전사개시점 -10 : Pribnow 상자 : (σ subunit 가결합 ) -35 : -35 영역 ; -35 ~ +1 (core promoter, 핵심촉진유전자 ) Figure 10.2 Sequence of representative promoters - E. coli

9 오페론 (operon) 원핵세포에서하나의염색체에서, 하나의 < 작동유전자 (operator)> 와이것에의해똑같이지배되는구조유전자들로이루어진유전자군. 오페론 (Operon) 의구성요소 : Operon: a group of operator, promoter, and structural genes that codes for proteins 1) 구조유전자 (structural genes): 단백질발현정보 2) 촉진유전자 (promoter) : 구조유전자의이웃에, 작동유전자와의사이에있으며, 구조유전자의작동개시점이되는유전자부분, RNA pol 결합 3) 작동유전자 (operator) : 촉진유전자근처에서촉진유전자의작용을조절하는유전자 (binds a activator or repressor protein) 4) 증진유전자 : 작동유전자근처에있으며, 그작동을조절하는유전자 (DNA) 부분으로제어물질 / 촉진물질 ( 억제유전자 / 촉진유전자 ) 을만든다.

10 Promoter structure in prokaryotes Promoter 5 PuPuPuPuPuPuPuPu AUG +1 [ ] mrna -35 region TTGACA AACTGT -36 T T G -31 A C A transcription start site T -10 region TATAAT ATATTA Pribnow box A T A A T 5 mrna % consensus sequences %

11 Operon 의전형적인구조 promoter Structural genes Operator (regulatory sequence that binds a activator or repressor protein) RNA transcript

12 대장균의 RNA 중합효소 (Prokaryotic RNA polymerase) RNA polymerase: 5 량체단백질 ( 분자량 : 약 5000,000 Da) Subunit Number Role a 2? b 1 phosphodiester bond 형성 b 1 DNA template 결합 s 1 promoter 인식 / 전사개시촉진 a 2 bb s a 2 bb + s holoenzyme core enzyme sigma factor ( 완전효소 ) ( 중심효소 ) 시그마인자 Termination factor rho (r ) : 전사종결에관여 4 량체단백질

13 sigma (s) factor 의기능 initiation factor RNA polymerase 가 promoter 에잘결합하도록도움. nonspecific binding (non-promoter DNA) : 불안정화 specific binding (promoter DNA) : 안정화 ( 올바른 promoter DNA 의탐색 ) Any DNA (nonspecific) K a (M -1 ) Promoter DNA (specific) Core 2 X Holo 1 X to 10 15

14 원핵세포의전사 (Prokaryotic Transcription) Figure 10.3 The order of events

15 RNA 합성의시작과종말점 Purine+4~5 nt 전사후분리 sigma re-cycled

16 RNA 합성 - S 도입 (topoisomerase 가 - supercoil 제거 ) (topoisomerase 가전사 / 복제시유발되는 + supercoil 제거 )

17 전사중생성되는초나선의해소 Figure 10.4 Topoisomerases remove supercoils

18 내제성종결인자에의한 Chain Termination Figure 10.5 Inverted repeats in DNA sequence

19 종결인자에의한 Chain Termination Figure 10.6 Termination by rho factor

20 Prokaryotic mrna 들은하나이상의단백질을한번에발현시킬수있다.

21 Prokaryotes: Tanscriptional Regulation 유전자의발현조절방법 : 증진유전자 (enhancers) : 전사인자결합부위전사활성 / 불활성인자 - 대체 s 인자 (alternative s factors):bacteria phage SPO1 SPO1 : 초기유전자가지고감염 Operons: 대장균의 repressor 에의한 Lac 오페론조절 (- regulation) activator 에의한조절 (+ regulation) : CAP 전사감쇠 (transcription attenuation) : 대장균의 trp 오페론조절

22 증진유전자 (enhancers) : 전사활성 / 불활성인자결합 대장균의 Fis enhancer site: 증진자결합 Fis : 전사증진인자 ( 단백질 )

23 대체 s 인자 (alternative s factors): viruses and bacteria produce different subunits that direct RNA polymerase to different genes. 박테리아유전자발현 바이러스유전자발현예 ) bacteria phage SPO1 박테리아 (B. substilus) 공격하는바이러스 바이러스시그마인자

Summary of Transcriptional Regulation - transcription is the primary control point for gene expression In all organisms but particularly in bacteria - control is most often effected by modulation of

24 Summary of Transcriptional Regulation - transcription is the primary control point for gene expression In all organisms but particularly in bacteria - control is most often effected by modulation of promoter activity Promoters can be turned on and off by binding regulatory proteins at sites near the promoter on the DNA [ near in prokaryotes ~ b.p. in eukaryotes ~ up to 50,000 b.p. typically <1000 b.p.] Positive Regulation: Binding of regulatory proteins increases transcription Negative Regulation: Binding of regulatory proteins reduces transcription

25 Modulation of promoter activity 1. Positive Regulation (activation): positive regulatory factor (activator) 가 RNAP 가잘작용하도록도움. Activator RNAP Activator binding site EXAMPLE: CAP/HSF

26 Modulation of promoter activity 2. Negative Regulation (repression): negative regulatory factor (repressor) 가작용해서 RNAP 가 promotor 에결합하는것을방지함. RNAP +1 Repressor Operator EXAMPLE: lac REPRESSOR

27 lac promoter 의조절 - P lac 목적 : 1) turn OFF (repress) P lac in the presence of glucose ( 포도당 ) 2) turn ON (induce or de-repress) P lac when lactose is the sole carbon source

28 NEGATIVE REGULATION INDUCIBLE TRANSCRIPTION The lac Operon REPRESSOR INDUCER X INACTIVE REPRESSOR X REPRESSOR BINDING SITE OPERATOR

29 Regulation of Gene Expression :Lac Operon *IPTG also induces ( 박테리아실험시사용 ) Splits lactose lactose transport??

30 pet28b

31 lac repressor : inducer 단백질발현유도 - + Lactose IPTG 실험실에서박테리아로단백질을과량발현시 RNAP 의전사를유도하기위해사용 (lactose:b-galactosidase 가분해 사용 x)

32 Enzymatic Activity of b-galactosidase 갈락토스글루코스 ( 포도당 )

33 CAP 과포도당 (glucose) : 연관성 (Catabolite activator protein) CAP 은포도당 sensor 이다. When [glucose] is low [camp] increases CAP is active in DNA binding When [glucose] is high, [camp] decreases CAP does not bind DNA When bound to DNA CAP activates transcription Transcription of lac operon is activated when [glucose] is low

34 Lac operon 의결합부위 Figure Binding sites in the lac operon

35 CAP mediates glucose repression of Lac Promotes transcription

36 Adenylate cyclase and CAP mediate glucose repression of Lac Adenylate cyclase (AC) is an enzyme that synthesizes cyclic AMP (camp) from ATP AC AMP glucose camp High glucose adenylate cyclase is inhibited (indirectly, via a catabolic product) Therefore camp levels are LOW Absence of glucose adenylate cyclase is NOT repressed Therefore camp levels are HIGH camp forms a complex with the CAP protein, which allows it to then bind to the CAP site upstream of the Lac operon. Binding of the CAP protein is required to allow RNA polymerase to bind to the lac promoter and turn on transcription. In the absence of CAP binding, there is no (or very little) transcription of the lactose operon, even in the presence of lactose.

37 Summary of lac operon regulation Lactose( 젖당 ) Glucose( 포도당 ) - + lac repressor RNAP CAP-cAMP + + (no: Weak)

38 Levels of Control of Lac Operon Expression 3 Scenarios: 1) No Lactose around Operon switched off, no mrna regardless of [glucose] 2) Lactose present; glucose also present The presence of lactose inactivates the repressor Transcription occurs (weak) Glucose present camp is low CRP does not help transcription 3) Lactose present; no glucose The presence of lactose inactivates the repressor Transcription occurs NO Glucose camp is high camp binds CRP (becomes activated) CRP binds & Helps Transcription High Level of transcription

39 THE trp OPERON Aporepressor Operator Active repressor Co-repressor X -In the presence of tryptophan in the cell, the genes are turned off. That is, mrna is not transcribed and the proteins are not made. -In the absence of tryptophan in the medium, the genes are turned on. That is, mrna is transcribed and proteins are made.

41 진핵세포 (Eukaryotes) 의전사 RNA 중합효소 I : 대부분의 rrna 전구체합성 RNA 중합효소 II : mrna 전구체합성 * RNA 중합효소 III : trna와 5S rrna 전구체합성 RNA 의전사후변형 세종류의 RNA (trna, rrna, mrna) 는모두전사후효소에의해변형되어각각의기능을갖는형태로된다.

42 진핵생물의유전자발현및변형 (Eukaryotic Gene Expression) Enhancer Promoter Transcribed Region Terminator Translation C N Polypeptide Transcription RNA Polymerase II Primary transcript 5 Intron1 3 7m G 7m G Exon1 Exon2 Cap Splice Cleave/Polyadenylate A n Transport A n

43 진핵세포 Yeast RNA polymerase II 의예

44 그림 효모 RNA 중합효소 Ⅱ 의구조.

45 그림 RNA Pol Ⅱ 프로모터의네가지요소들.

47 진핵세포의전사의예

48 그림 DNA 와결합한효모 TATA- 결합단백질 (TBP) 의모델.

49 DNA /RNA 결합구조 (domain /motif) Helix-turn-helix motif 그림 헬릭스 - 턴 - 헬릭스모티프.

50 그림 Cys His 징크 - 핑거모티프. 2 2 Zinc finger motif

51 Leucine zipper motif 그림 염기성지역류신지퍼를갖고있는 DNA- 결합단백질

52 mrna processing mrna Processing ( 외부핵산가수분해효소로부터보호 ) 5 -capping 3 -poly adenylation Intron 제거 +splicing ( 액손연결 ) 5 -capping ( 갓구조생성 : 전사와동시 ) 7-methylguanosine 5-5 : 삼인산결합 2 OH : methylation mrna 안정화에기여 3 -poly adenylation 100~ 200 nucleotides (mrna 가핵을떠나기전에붙여짐.)

53 진핵생물 mrna 는 3 poly(a) tail 을갖게된다. polyadenylation catalyzed by poly(a) polymerase

54 5 -capping 5 -capping ( 갓구조생성 : 전사와동시 ) 7-methylguanosine 5-5 : 삼인산결합 2 OH : methylation mrna 안정화에기여

55 진핵생물의 DNA 와 mrna 는 intron 들이있고, mrna 의 intron 들은추후에제거된다. 진핵생물 :Coding sequence gene 은연속적이아니다.

56 mrna degradation The rate of degradation of mrna vary greatly Average half-life is about 3 hrs Degraded by ribonuclease (RNase) Stable mrnas carry a sequence at 3 ends that inhibit RNase Poly(A) tail is important to the stability Major degradation pathway shortening poly(a) tail decapping 5 end degrading RNA in 5 3 direction

57 Intron 의제거 (mrna splicing)

58 Secondary structure trna 의구조 CCA Tertiary structure 모든 trna 는 3 -end 에 CCA 순서가존재 : 아미노산부착

59 trna processing

60 splicing 리보자임 (Ribozyme) : 효소역활의 RNA Group I (Self-splicing intron that requires an external guanine-containing nucleotide for splicing; releases the intron in a linear form.) intracellular guanosine residue 3 -OH is attacking group linear form circular Group II (Self-splicing intron that does not require an external nucleotide for splicing; releases the intron in a lariat form.) 2 -OH adenosine within intron 올가미구조 (Lariat form) Spliceosome catalyzed largest group same as group II except that it needs snrnps small nuclear ribonucleoproteins each snrnp contains snrnas snrnas : U1, U2, U3, U5, U6

61 Group I intron O O OH O= P - O - G O- U - O - P - O - O- P - O - A O- exon Cyclized intron

62 Group II intron Branching point adenosine 2 -OH Exon : 올가미구조 Lariat

63 스플리이소솜 (Spliceosome) (50S ~ 60S 크기 ) 핵에서 pre-mrna 의 splicing 에관여. 작은리보핵산단백질 (snrnps: 스넙스 ) 와 RNA (U1 ~ U5) 가관여.

Chapter 26

Chapter 26 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