本文選題：布氏錐蟲 + PWWP結(jié)構(gòu)域��； 參考：《中國科學(xué)技術(shù)大學(xué)》2016年博士論文

【摘要】：基因轉(zhuǎn)錄的正常進行對細胞的生命活動非常重要,幾十年來一直是研究的熱點。高等真核生物的轉(zhuǎn)錄過程已經(jīng)得到比較詳盡的研究,但一些單細胞真核生物比如布氏錐蟲的轉(zhuǎn)錄過程了解還較少。由于布氏錐蟲較早從進化樹中分枝出來,其轉(zhuǎn)錄過程具有非常獨特的特征,比如其：mRNA是以多順反子形式轉(zhuǎn)錄形成的,另外許多轉(zhuǎn)錄因子是缺失的。在本篇論文中,我們主要對布氏錐蟲轉(zhuǎn)錄延伸因子TFIIS和PAF1復(fù)合體的結(jié)構(gòu)和功能進行了研究。TFIIS是一類比較保守的轉(zhuǎn)錄因子,它能夠使轉(zhuǎn)錄受到阻滯的聚合酶重新恢復(fù)轉(zhuǎn)錄活性,大大提高轉(zhuǎn)錄效率,在真核生物中研究的比較清楚。在布氏錐蟲中,目前已鑒定出三個,分別命名為TbTFIIS1, TbTFIIS2-1和TbTFIIS2-2但TFIIS在布氏錐蟲轉(zhuǎn)錄調(diào)控中的作用目前還不清楚。我們通過RNA干擾實驗發(fā)現(xiàn),當(dāng)單獨敲除TFIIS2-1或TFIIS2-2時,不影響細胞生長,但同時敲除TFIIS2-1和TFIIS2-2時,細胞的生長受到明顯抑制,這說明在布氏錐蟲中TFIIS2-1和TFIIS2-2的功能可能是冗余的。有趣的是,通過序列比對我們發(fā)現(xiàn),與其他物種TFIIS不同的是,TbTFIIS2-1和TbTFIIS2-2的N端各具有一個PWWP (TFIIS2-1 PWWP和TFIIS2-2 PWWP)結(jié)構(gòu)域,這也是第一次在轉(zhuǎn)錄因子中發(fā)現(xiàn)PWWP結(jié)構(gòu)域。我們通過核磁方法分別解析了這兩個PWWP結(jié)構(gòu)域的結(jié)構(gòu)。這兩個PWWP結(jié)構(gòu)域的結(jié)構(gòu)與其它PWWP結(jié)構(gòu)域結(jié)構(gòu)類似,都具有典型的PWWP折疊模式,包括一個由五條反平行β片層組成的β桶和一個α螺旋。之前研究發(fā)現(xiàn)PWWP結(jié)構(gòu)域具有識別甲基化組蛋白和DNA的能力。我們分別用AT-、GC-rich ssDNA和dsDNA序列進行了化學(xué)位移微擾實驗,發(fā)現(xiàn)TFIIS2-1 PWWP和TFIIS2-2 PWWP結(jié)構(gòu)域都不具有結(jié)合DNA的能力。另外也通過核磁滴定發(fā)現(xiàn)TFIIS2-2 PWWP結(jié)構(gòu)域通過一個保守的芳香族籠子特異性地識別H4K17me3和H3K32me3,這種結(jié)合模式與其他PWWP結(jié)構(gòu)域相似,而TFIIS2-1 PWWP結(jié)構(gòu)域則不結(jié)合甲基化小肽。通過對TFIIS2-1 PWWP和TFIIS2-2 PWWP結(jié)構(gòu)域進行序列比對發(fā)現(xiàn),形成保守籠子的三個芳香族氨基酸對PWWP結(jié)構(gòu)域結(jié)合甲基化組蛋白是必須的。TFIIS2-2 PWWP結(jié)構(gòu)域是第一個在布氏錐蟲中發(fā)現(xiàn)的能結(jié)合甲基化組蛋白的PWWP結(jié)構(gòu)域,將為研究布氏錐蟲的轉(zhuǎn)錄提供一定的理論基礎(chǔ)。我們進一步通過串聯(lián)親和純化發(fā)現(xiàn),在布氏錐蟲中,轉(zhuǎn)錄延伸因子TFIIS與PAF1復(fù)合體存在相互作用。PAF1復(fù)合體在真核生物中比較保守,由PAF1,RTF1, CTR9, LEO1和CDC73五個亞基組成,對許多生命活動,比如基因表達與沉默、DNA修復(fù)以及細胞周期調(diào)控等非常重要。已有文獻報道,布氏錐蟲的PAF1復(fù)合體組成亞基有CTR9, LEO1和CDC73,由于序列同源性太低,布氏錐蟲中是否存在PAF1和RTF1亞基,目前還是未知。在本篇論文中,我們通過生物信息學(xué)分析和串聯(lián)親和純化實驗,確認兩個不含有特殊結(jié)構(gòu)域的未知功能蛋白Tb927.3.5070和Tb927.7.4030分別為布氏錐蟲的PAF1和RTF1亞基,并通過酵母雙雜交確認了各個亞基的相互作用網(wǎng)絡(luò)。通過RNA干擾實驗發(fā)現(xiàn),PAF1復(fù)合體對細胞的生長是必須的。同時,我們通過GST pull down實驗證明,TFIIS2-1中domain I(LW結(jié)構(gòu)域)和TFIIS2-2 C末端的LW結(jié)構(gòu)域直接參與LEO1的相互作用。高通量測序結(jié)果顯示,PAF1復(fù)合體不僅參與基因的轉(zhuǎn)錄而且也參與到布氏錐蟲VSGES沉默的調(diào)控,通過實時熒光定量PCR實驗也驗證了這一結(jié)果。另外,酵母雙雜交實驗表明,PAF1復(fù)合體與ISWI直接相互作用,我們推測其可能通過ISWI來參與VSGES沉默。
[Abstract]:The normal progression of gene transcription is very important for cell life activities and has been the focus of research for several decades. The transcriptional process of higher eukaryotes has been studied in more detail, but some single cell eukaryotes, such as Trypanosoma brucellae, have less understanding of the transcription process. The transcriptional process has very unique characteristics, such as: mRNA is transcribed in the form of polycometon, and many other transcription factors are missing. In this paper, we mainly study the structure and function of TFIIS and PAF1 complexes of Trypanosoma brucellus, which are a kind of relatively conservative transcription factors. It can reactivate transcriptional transcriptional reactivity and greatly improve transcriptional efficiency. It is clearly studied in eukaryotes. In Trypanosoma brucellus, three are identified, named TbTFIIS1, TbTFIIS2-1 and TbTFIIS2-2, but the role of TFIIS in the transcription regulation of Trypanosoma brucellus is not yet clear. RNA interference experiments showed that the cell growth was not affected when TFIIS2-1 or TFIIS2-2 was knocked out alone, but when TFIIS2-1 and TFIIS2-2 were knocked out, the cell growth was significantly inhibited, which suggests that the function of TFIIS2-1 and TFIIS2-2 in Trypanosoma brucellus may be redundant. The same is that the N end of TbTFIIS2-1 and TbTFIIS2-2 has a PWWP (TFIIS2-1 PWWP and TFIIS2-2 PWWP) domain, which is the first time to find the PWWP domain in the transcription factor. We parsed the structure of the two PWWP domains by NMR. The structure of the two PWWP domains is similar to that of other PWWP domains. A typical PWWP folding pattern, including a beta bucket and an alpha helix composed of five anti parallel beta lamellae, has been found to have the ability to identify methylation histone and DNA in the PWWP domain. We used AT-, GC-rich ssDNA and dsDNA sequences to perform chemical shift perturbation experiments, and found TFIIS2-1 PWWP and TFIIS2-2 PWWP junction. The domain does not have the ability to combine DNA. In addition, the TFIIS2-2 PWWP domain identifies H4K17me3 and H3K32me3 specifically through a conservative aromatic cage, which is similar to other PWWP domains, while the TFIIS2-1 PWWP domain is not combined with the methylation small peptides. Sequence alignment of the PWWP domain found that three aromatic amino acids formed in the conservative cage were the necessary.TFIIS2-2 PWWP domain of the PWWP domain binding methylation histone, the first PWWP domain of the binding methylation histone found in Trypanosoma brucelli, which would provide a theoretical basis for the study of the transcription of Trypanosoma brucellus. We further found that in Trypanosoma brucellus, the interaction of transcription extension factor TFIIS and PAF1 complex in Trypanosoma brucellus is more conservative in eukaryotes, consisting of five subunits of PAF1, RTF1, CTR9, LEO1 and CDC73, for many life activities, such as gene expression and silence, DNA repair and cell cycle. Regulation and so on is very important. It has been reported that the subunits of the PAF1 complex of Trypanosoma brucellus are CTR9, LEO1 and CDC73. As the sequence homology is too low, the existence of PAF1 and RTF1 subunits in Trypanosoma brucellus is still unknown. In this paper, we have confirmed that two non specific compounds were not contained by bioinformatics analysis and tandem affinity purification experiments. The unknown functional proteins Tb927.3.5070 and Tb927.7.4030 are PAF1 and RTF1 subunits of Trypanosoma brucellae, respectively, and the interaction network of each subunit is confirmed by yeast two hybrid. The RNA interference experiment shows that the PAF1 complex is necessary for the growth of the cells. At the same time, we have proved by the GST pull down experiment that TFIIS2-1 do is in do. The LW domains at the end of the main I (LW domain) and TFIIS2-2 C are directly involved in the interaction of LEO1. High throughput sequencing results show that the PAF1 complex not only participates in gene transcription but also participates in the regulation of the VSGES silencing of Trypanosoma brucellae, and the results are also verified by real time fluorescence quantitative PCR experiments. In addition, yeast two hybrid experiments show that PAF The 1 complex directly interacts with ISWI, and we speculate that it may participate in VSGES silencing through ISWI.

【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q78
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本文編號：1800632