Chapter 15 RNA Polymerase II: Basal Transcription 1
Eukaryotic and bacterial RNA polymerases differ in some aspects 1. eukaryotes have three RNA polymerases, with one each specifically dedicated to rrna, mrna, and trna synthesis bacteria have a single RNA polymerase 2. eukaryotic RNA polymerases require numerous transcription factors bacterial RNA polymerases require one or two at most 3. eukaryotic RNA polymerase requires factors to modify chromatin structure to allow RNA polymerase access bacterial genes are fully accessible and tend to be active or require only one or two factors to become active 4. eukaryotes process mrnas by capping, polyadenylation, and splicing bacterial RNAs do not have introns and the only modification of transcripts is polyadenylation to mark the mrna for degradation 2
15.1 Introduction to RNA polymerase II 진핵세포의핵에서 3 가지의 RNA polymerase 가발견됨. RNA polymerase I, II, III 는아래표에서보듯이각각다른성격을갖음 α-amanitin 에대한민감도가달라농도를조절하여반응을조절함. ( 교과서그림 15.3 참조 ) actinomycin D 에대하여도다른민감도를갖음. 이항생제는 DNA 의 C-G, G-C 염기쌍에결합하여가닥의분리를막음. rrna 유전자에 GC 가많아민감하게작용. ( 교과서그림 15.4 참조 ) 3
Eukaryotic RNA polymerases are more complex than prokaryotic ones RNA polymerase I has 14 subunits RNA polymerase II has 12 subunits RNA polymerase III has 17 subunits Bacterial RNA polymerase has 5 subunits Archaeal RNA polymerase 11 subunits The genes encoding the RNA polymerase II subunits are essential; Deletion mutants for RPB4 and RPB9 are conditional mutants; deletion of all other RNA polymerase II subunit genes are nonviable. 4
Eukaryotic RNA polymerases share some subunits but not others Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12 are present in RNA polymerases I, II, and III ( 진핵세포의 RNA polymerase 간에일부 subunit 를 share 함. 그림 15.5 에는생략됨 ) Many RNA polymerase II subunits have homologs in RNA polymerase I and/or III The prokaryotic and eukaryotic RNA polymerases exhibit homologies archaea and bacteria have a single RNA polymerase to synthesize mrna, rrna, and trna Figure 15.05 the bacterial core RNA polymerase subunits each has at least one homolog in each eukaryotic nuclear RNA polymerase 5
15.2 RNA polymerase IIstructure Kornberg 는 yeast RNA polymerase 에서 Rpb4 와 Rpb7 subunit 가빠진구조의결정을만듬 Figure 15.6 Structure of the Δ4/7 RNA polymerase II Eukaryotic and prokaryotic RNA polymerases have similar core structures 효모와 Thermus aquaticus RNA polymeras 의유사성비교 ( 좌 ) 서열의유사성 ( 유사부위가붉은색으로표시됨 ) ( 우 ) 구조의유사성 ( 유사부위가녹색으로표시됨 ) active site 가있는 cleft 부위가유사성이많음. 6
The structure of the 12 subunit RNA polymerase II was finally solved RNA polymerase 의최종구조 RNA polymerase II transcription elongation complex 7
The roles of fork loop 1, rudder, and lid in transcription elongation 7-8 base pair 로된 DNA-RNA hybrid 는 active site 로부터나오면서방향이바뀌면서분리가되어나온다. clamp 로부터돌출된세개의 loop 인 lid, rudder, fork loop 1 이 hybrid dissociation, RNA exit, maintenance of the upstream end of the transcription bubble 에중요한역할을한다. fork loop 1 prevents premature unwinding of the DNA-RNA hybrid lid acts as a wedge to separate the DNA and RNA strands and guide the RNA to the exit path the rudder prevents the DNA from reannealing with the RNA strand 8
The bridge helix acts as a ratchet to advance RNA polymerase on DNA RNA polymerase가새로운염기를가하면서 DNA 주형을따라움직이는가를설명함. Rpb1과 Rpb2 사이에만들어진 cleft에걸쳐있는 Rpb1 subunit가 bridge helix로작용하며 straight 구조와 bent 구조사이에서변화하면서진행을시킴. ( 좌 ) synthesis; nucleotide triphosphate (NTP) 가비워있는부위를채워 RNA 가닥의 3 을연결한다. (A는 active site. 핑크점은 magnesium ion). Translocation; nucleic acid가이동을하며 helix가 straight ( 회색 ) 에서 bent ( 보라색 ) conformation으로변한다. Relaxation; bridge helix가다시 straight ( 회색 ) 로변하면서 NTP가첨부될수있는부위가생김 9
15.3 Transcription initiation site identification RNA polymerase 는전사를시작하기위하여서 transcription factor 의도움이필요하다. 그과정을 15 장에서배우고 RNA polymerase I 과 III 에대하여서는 18 장에서배운다. RNA polymerase II transcription begins at specific initiation sites RNA polymerase 는특정한부위에서전사를시작한다. 전사시작부위를찾는방법에는 3 가지가있는데 S1 mapping (S1 nuclease analysis), primer extension, run-off analysis 가있으며앞의두가지는 living cell 과 cell free-system 에서사용가능하고마지막방법은 cell-free system 에서사용가능하다. S1 Mapping 방사성동위원소로표시된 DNA probe 를세포에서추출한 RNA 와결합을시킨다. 결합후 S1 nuclease 로단일가닥을절제하고남은 DNA-RNA hybrid 를 denature 시킨다. 이곳에남은 DNA 를전기영동을하여크기를파악한다. 그림에서는 275 base 이고그로인하여시작부위를찾을수있다. Figure 15.12 10
Primer extension ( 좌 ) 전사시작부위로부터 50-150 nucleotide downstream 부위의염기서열을갖은 18 nucleotide 의크기의 DNA primer 를합성한다. 이를 RNA 와교잡한후 reverse transcriptase 로 5 말단을합성한다. 변성시킨후전기영동을이용하여시작부위를파악 Run-off transcription Figure 15.13 ( 우 ) 관심이있는유전자 (light blue) 를 plasmid에넣은후전사시작부위에서약 200 nucleotide downstream 부위를제한효소를이용하여자른다. 이곳에 radioactive nucleoside triphosphate, RNA polymerase 등을첨가하여전사를야기시킨후 RNA 전사체가만들어지면전기영동을하여크기를 11 밝혀낸다.
Many genes can be monitored simultaneously by DNA microarrays ( 좌 ) DNA 의발현을보기위하여 micro array 를이용한다. 6 주된 hypothyroid mice 와 thyroxine 을주입한동일한연령의쥐로부터간을추출한후 RNA 를분리하여 cdna 를만든다. hypothyroid liver 에분리한 cdna 는 green 으로 label 하고 hyperthyroid liver 에서분리한 cdna 는 red 로 label 한다. cdna probe 를 mix 한후동시에 microarray 에 hybridization 시킨다, green 은 hypothyroid liver 에서많이만들어지는 RNA 이고 red 는반대임. yellow 는관계없이양쪽에서만들어지는 RNA 를나타냄. ( 우 ) reporter gene를만들어서 regulatory region의 effect를살펴보는실험. 관심있는유전자의 regulatory 지역을 coding sequence가알려진유전자의앞부분에삽입을한다. 만들어진 recombinant DNA를진핵세포내에삽입한후 reporter 단백질의합성에 regulatory region이어떠한 effect를미치는가를조사함. 외부 DNA를진핵세포내로넣는것을 transfection이라고한다. 12