布氏锥虫转录延伸因子TFIIS和PAF1复合体的结构与功能研究

发布时间：2018-04-25 08:51

  本文选题：布氏锥虫 + PWWP结构域　； 参考：《中国科学技术大学》2016年博士论文

【摘要】：基因转录的正常进行对细胞的生命活动非常重要,几十年来一直是研究的热点。高等真核生物的转录过程已经得到比较详尽的研究,但一些单细胞真核生物比如布氏锥虫的转录过程了解还较少。由于布氏锥虫较早从进化树中分枝出来,其转录过程具有非常独特的特征,比如其：mRNA是以多顺反子形式转录形成的,另外许多转录因子是缺失的。在本篇论文中,我们主要对布氏锥虫转录延伸因子TFIIS和PAF1复合体的结构和功能进行了研究。TFIIS是一类比较保守的转录因子,它能够使转录受到阻滞的聚合酶重新恢复转录活性,大大提高转录效率,在真核生物中研究的比较清楚。在布氏锥虫中,目前已鉴定出三个,分别命名为TbTFIIS1, TbTFIIS2-1和TbTFIIS2-2但TFIIS在布氏锥虫转录调控中的作用目前还不清楚。我们通过RNA干扰实验发现,当单独敲除TFIIS2-1或TFIIS2-2时,不影响细胞生长,但同时敲除TFIIS2-1和TFIIS2-2时,细胞的生长受到明显抑制,这说明在布氏锥虫中TFIIS2-1和TFIIS2-2的功能可能是冗余的。有趣的是,通过序列比对我们发现,与其他物种TFIIS不同的是,TbTFIIS2-1和TbTFIIS2-2的N端各具有一个PWWP (TFIIS2-1 PWWP和TFIIS2-2 PWWP)结构域,这也是第一次在转录因子中发现PWWP结构域。我们通过核磁方法分别解析了这两个PWWP结构域的结构。这两个PWWP结构域的结构与其它PWWP结构域结构类似,都具有典型的PWWP折叠模式,包括一个由五条反平行β片层组成的β桶和一个α螺旋。之前研究发现PWWP结构域具有识别甲基化组蛋白和DNA的能力。我们分别用AT-、GC-rich ssDNA和dsDNA序列进行了化学位移微扰实验,发现TFIIS2-1 PWWP和TFIIS2-2 PWWP结构域都不具有结合DNA的能力。另外也通过核磁滴定发现TFIIS2-2 PWWP结构域通过一个保守的芳香族笼子特异性地识别H4K17me3和H3K32me3,这种结合模式与其他PWWP结构域相似,而TFIIS2-1 PWWP结构域则不结合甲基化小肽。通过对TFIIS2-1 PWWP和TFIIS2-2 PWWP结构域进行序列比对发现,形成保守笼子的三个芳香族氨基酸对PWWP结构域结合甲基化组蛋白是必须的。TFIIS2-2 PWWP结构域是第一个在布氏锥虫中发现的能结合甲基化组蛋白的PWWP结构域,将为研究布氏锥虫的转录提供一定的理论基础。我们进一步通过串联亲和纯化发现,在布氏锥虫中,转录延伸因子TFIIS与PAF1复合体存在相互作用。PAF1复合体在真核生物中比较保守,由PAF1,RTF1, CTR9, LEO1和CDC73五个亚基组成,对许多生命活动,比如基因表达与沉默、DNA修复以及细胞周期调控等非常重要。已有文献报道,布氏锥虫的PAF1复合体组成亚基有CTR9, LEO1和CDC73,由于序列同源性太低,布氏锥虫中是否存在PAF1和RTF1亚基,目前还是未知。在本篇论文中,我们通过生物信息学分析和串联亲和纯化实验,确认两个不含有特殊结构域的未知功能蛋白Tb927.3.5070和Tb927.7.4030分别为布氏锥虫的PAF1和RTF1亚基,并通过酵母双杂交确认了各个亚基的相互作用网络。通过RNA干扰实验发现,PAF1复合体对细胞的生长是必须的。同时,我们通过GST pull down实验证明,TFIIS2-1中domain I(LW结构域)和TFIIS2-2 C末端的LW结构域直接参与LEO1的相互作用。高通量测序结果显示,PAF1复合体不仅参与基因的转录而且也参与到布氏锥虫VSGES沉默的调控,通过实时荧光定量PCR实验也验证了这一结果。另外,酵母双杂交实验表明,PAF1复合体与ISWI直接相互作用,我们推测其可能通过ISWI来参与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.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78
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本文编号：1800632