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作者(中文):林新閎
作者(外文):Lin, Hsin-Hung
論文名稱(中文):RNA聚合酶II中轉錄因子TFIIF的冷凍電顯結構
論文名稱(外文):Cryo-EM structure of transcription factor TFIIF in RNA polymerase II
指導教授(中文):章為皓
林俊成
指導教授(外文):CHANG, WEI-HAU
LIN, CHUN-CHENG
口試委員(中文):陳宏達
杜憶萍
鄒德里
鍾思齊
口試委員(外文):Chen, Hung-Ta
Tu, I-Ping
Tzou, Der-Lii M.
Chung, Szu-Chi
學位類別:博士
校院名稱:國立清華大學
系所名稱:化學系
學號:103023458
出版年(民國):111
畢業學年度:110
語文別:英文
論文頁數:114
中文關鍵詞:RNA聚合酶(RNAP-II)轉錄因子IIF(TFIIF)低溫冷凍電子顯微鏡單分子螢光共振能量轉移
外文關鍵詞:RNA polymerase IITranscription Factor IIF (TFIIF)Cryo-electron microscopy (Cryo-EM)single molecule fluorescence resonance energy transfer (smFRET)
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由RNA聚合酶II(RNAP-II)所執行的DNA轉錄是一個多重步驟的過程。在起始步驟,RNAP-II通過與轉錄因子的結合去辨識出基因序列中轉錄的起始點 ,而過程中,普遍存在於真核細胞中的通用轉錄因子IIF(TFIIF) 參與了轉錄起始的輔助,並會在起始結束後繼續輔助RNA聚合轉錄的延伸步驟,甚至涉及到轉錄反應的中止步驟。轉錄因子透過結合於RNAP-II上對應的位置達成轉錄的調控。因此,由Tfg1以及Tfg2兩個次單元所構成的TFIIF,以其二聚體區域和次單元兩末端區域向RNAP-II形成複合體。然而,在先前X光繞射晶體的結構研究中,高解析度的電子雲圖(4埃)未能顯現這樣的複合形式。即便後來透過低溫冷凍電子顯微鏡(cryo-EM)獲得的起始複合體結構中,也僅定位了二聚體區域與RNAP-II額葉區(Lobe)的空間關係。但對於延伸態複合體,現有的結構資訊仍不能描繪出TFIIF各區域與RNAP-II的結合形式。
在本篇研究中,我們透過低溫冷凍電子顯微鏡(cryo-EM)的技術,解出了RNAP-II與TFIIF延伸態複合體的高解析度(2.11埃)結構。並藉由3D影像分類計算,將TFIIF與RNAP-II的複合暫態進行結構拆分,最終獲得了多種不同型態的結合模式。其中,我們發現TFIIF中Tfg2的翼狀結構區(Tfg2 WH)不但會如同起始態般,與RNAP-II的DNA出入口作用,也會換位到RNAP-II次單元Rpb10上形的全新結合位。最後,我們用單分子螢光共振能量轉移(smFRET)測試我們cryo-EM的動態模型,證實了Tfg2 WH的確能在上述位置間做出轉換。這個在轉錄延伸態中首次發現的TFIIF的換位行為,預測了它如何巧妙地避免與其他輔助因子(如Spt5)的衝突,並在延伸結束後,能再轉換回起始態的位置,開起下一輪的轉錄。
Transcription Factor II F (TFIIF) is one of essential cofactors for RNA polymerase II (RNAP-II), including in the group of general transcription factors (GTF). It mainly composed of two subunits (Tfg1 and Tfg2), and has been found to be a conserved factor in eukaryotes. Regarding the function of TFIIF, TFIIF has been reported to play roles in both initiation and elongation phases of RNA synthesis, and speculated to be involved in the termination of RNAP-II. Those complex activities are evidently connected to the structural information and conformational changes of protein complex. However, the structure of TFIIF was partially answered only when formed the initiation complexes with other GTFs. The architecture about how TFIIF interacts with RNAP-II during elongation is still unclear. Here, by using the method of cryo-EM and 3D computational analysis, a high resolution (2.11A) map of elongating RNAP-II was solved as the basis, and the binding situation of TFIIF can be classified into multiple groups. In this study, an alternative binding position for the winged helix domain of Tfg2 (Tfg2 WH) on Rpb10 of RNAP-II was confirmed in one of reconstructed group, and the molecular model based on this cryo-EM map suggests the candidates of amino acids in the binding interface between subunits. Furthermore, by combing the cryo-EM results with the measurement of single molecule fluorescence resonance energy transfer (smFRET), the mobility of Tfg2 WH was demonstrated with a great transposition leaving from the hotspot of transcription. The finding of mobility may be related to the multiple function of TFIIF and provides a possibility for TFIIF to co-work with other elongation factors (such as spt5).
Table of Contents
Abstract------------------------------------------------------------1
摘要-----------------------------------------------------------------2
Acknowledgement-----------------------------------------------------3
List of Abbreviation------------------------------------------------4
List of Figure------------------------------------------------------6
List of Table-------------------------------------------------------7
1. Introduction-----------------------------------------------10
1.1. Role of RNA polymerase II in Eukaryotic gene expression----10
1.1.1. Transcription is a process producing RNA based on DNA code-10
1.1.2. Three types of RNA polymerase in eukaryotic cells----------11
1.1.3. RNA polymerase II is a 12-subunits protein complex---------11
1.1.4. Transcription Factors--------------------------------------14
1.1.5. Transcription Cycle of RNA polymerase II-------------------14
1.1.6. Transcription Factor IIF as a dual function factor---------15
1.1.7. TFIIF domains and their positioning in RNAP-II initiation complex------------------------------------------------------------16
1.1.8. Inferring TFIIF on RNAP-II in elongation-------------------19
1.1.9. Possible causes--------------------------------------------21
1.2. Cryogenic Electron Microscopy for Structure Analysis-------22
1.2.1. Cryo-EM resolution revolution------------------------------22
1.2.2. Analyzing TFIIF on RNAP-II as identifying its small or dynamic domains in a large protein complex---------------------------------25
1.2.3. Sub-ensemble analysis of protein complex via 3D Variability Analysis-----------------------------------------------------------26
2. Result-----------------------------------------------------28
2.1. Complex formation and Cryo-EM session----------------------28
2.2. Consensus model refinement and 3D variance analysis--------31
2.3. Feature of TFIIF - Dimerization Domain---------------------39
2.4. Saccharomyces-specific N-terminal domain in Tfg1-----------44
2.5. TFIIF support the stability of scaffold--------------------49
2.6. Wing Helix Domain of Tfg2 touch to Rpb10-------------------52
2.7. Tfg2 WH-domain vibrating at cleft--------------------------58
3. Discussion-------------------------------------------------63
3.1. Structural polymorphism in the cryo-EM dataset-------------63
3.2. Electrostatic complementarity at the interface-------------64
3.3. Tfg2 WH on Rpb10-------------------------------------------65
3.4. Evaluation of the location of Tfg2 WH by smFRET------------68
3.5. The mobility of Tfg2-WH------------------------------------73
4. Supplementary----------------------------------------------77
5. Reference--------------------------------------------------84
6. Appendix -- Dealing with High-Throughput cryo-EM Data-----95
6.1. Evolution of Hardware - Automatic Data Collection----------95
6.1.1. Camera (Detector)------------------------------------------95
6.1.2. TEM Machine------------------------------------------------99
6.1.3. Auto-system-----------------------------------------------100
6.1.4. Summary for evolution of hardware-------------------------102
6.2. Optimization of Data Flow - Transfer and Storage----------102
6.3. Evolution of Software – Algorithm-------------------------103
6.3.1. Friendly GUI----------------------------------------------104
6.3.2. Introduce Statistical Estimation to overcome the noise----104
6.3.3. GPU Acceleration------------------------------------------105
6.3.4. Reference Free Reconstruction-----------------------------106
6.3.5. 3D volume analysis----------------------------------------108
6.3.6. Machine Learning in cryo-EM field-------------------------109
6.4. Optimization of Workflow - on-the-fly processing----------110
6.5. Future Prospects------------------------------------------111
7. Reference for Appendix------------------------------------113

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