By 林書恒 DNA/RNA 的基本資訊 一、 DNA 與 RNA 的差別：2’碳 3’-deoxyadenosine：3’碳的-OH→-H →terminate transcription or translation DNA replication ~800 bp/sec Protein translation ~15 aa/sec RNA Transcription ~40 nt/sec T7 RNA polymerase ~300 nt/s Phosphate α,β,γ →去 β,γ，以 α 相接 NTP： ：nucleatid base pair
二、
E. coli polymerase
Movement model： Sliding Inchworm→像毛毛蟲一樣的前進，POLYMERASE 非此模式
Stages of transcription: stage
狀態
σ factor
closed complex (close binary
duplex DNA
→N-terminal region block DNA-binding region in
complex)
holoenzyme →σ factor recongnization
open complex (open binary complex)
Unwinding the DNA helix (Tx bubble) (about 12~14 bp in length)
tertiary complex
→ polymerase+DNA+RNA →RNA-DNA hybrid 約 8~9 bp.
Abortive initiation
→約 2~12 a.a. →A certain probility that the enzyme will release the RNA chain →之後重新從+1 開始
→分離 N-terminal region →DNA bend(同時 melting)以 達到 active site
→此時σ factor 仍皆在 promoter 上 →若有錯誤的 mRNA 產生，σ factor 會作為 obstacle 將 mRNA abortive →又稱 promoter clearance →失去σ factor ，complex covering region 變小
Release of σ factor(RNA synthesis begins) Bubble moves on
Prokaryotes have a single RNA polymerase Eukaryotes RNA Pol
I
rRNA
RNA Pol
II
mRNA
RNA Pol
III
tRNA, 5S rRNA
2α
rpoA
Ensyme assembly Promoter recognition→2 C-Terminal Domain(2CTD) →up element
β
rpoB
Catalytic Center
β’
rpoC
Catalytic Center Template-binding
σ
rpoD
Promoter specificity
ω Core=2α+β+β’+ω Holoenzyme=core+σ σ Cycle of making and breaking bonds between enzyme and nucleic acids →nucleatide addition 與 pol 的形狀改變相關，enzyme 對 growing chain 與 template 的鍵結，隨 nucleatid 而形成 cycle
About 7000 RNA polymerase molecules are present in an E.. coli cell 1. α subunit ADP-ribosylation on an arginine upon T4 infection
2. β subunit Rifampicin
→bind to the β subunit →inhibit transcription initiation. →Blocking the path for extending RNA chain →Mutation in rpoB gene can result in rifampicin resistance
Streptolydigins
→Inhibits transcription elongation →bind to the β subunit →Mutation in rpoB gene can result in resistance
→β subunit may contain two domains responsible for
initiation and
elongation 3. β’ subunit Heparin: →binds to theβ’ subunit →inhibits transcription in vitro →competes with DNA ∴β’ subunit may be responsible for binding to the template DNA . 4. σ factor： ： σ factor is released from the RNA pol after initiation (RNA chain is 8-9 nt) Holoenzyme has ~104-fold lower affinity for loose binding Holoenzyme has ~103-fold higher affinity for specific binding Totally, σ factor can result in 107 increase
Mg2+的參與
Polymerase 找 sequence: Directed walk
三、
Promoter →cis-acting site
-35 hexamer 多為 TTGACA，與-10 間的 spacer 多為~16nt -10 hexamer =TATA box=Pribnow box 多 TATAAT Up element 為-35 前~20nt，A/T content 高(A-T rich)
Promoter 為 consensus sequence， The sequence of the first 30 bases to be transcribed controls the rate at which the RNA polymerase clears the promoter Some promoter sequence are not strong enough →activating factor is required Supercoiling →overwound (positive supercoiling) →unwound (negative supercoiling) Interaction region
→研究方法： modification 1. DNA 可事先 modification (premodification) →又稱 touch down，可得知 essential position 2. Modification of RNA polymerase-DNA complex →得到 DNA footprinting 四、
Termination 1. 無 Rho →hairpin(G/C rich)+U-rich sequence 2. 有 Rho →亦須 hairpin →發現：in vitro 需添加 Rho (又稱 Rho dependent termination) In vivo 中以 mutation 證明其影響 →hexamer →ATP-dependent helicase →binding at rut site →受 nut site 調控(藉由接上 antitermination complex 抑制 Rho action) →受 ribosome 影響，ribosome 會阻止 Rho 前進
使 termination 失效的 ancillary factor →antitermination →readthrough