Topology of Archaeal Transcription Pre-Initiation Complex

斯科特9631093生物物理和生化方法将被用来研究超高温考古菌的转录预起始复合体的结构和蛋白质和DNA组分的组装。单个古生物转录系统与真核生物的同源性高于细菌转录系统，这表明古生物祖先系统分别使用RNA聚合酶(RNAP)I、II和III进化成三个真核生物转录复合体，合成rRNA、mRNA和tRNA。对古生菌基础转录的分子拓扑和机制的理解有望有助于揭示真核转录，并提供与三种真核系统分化相关的进化信息。之所以选择P.Furiosus转录系统进行研究，是因为：(A)人们对古生菌转录及其调控知之甚少；(B)古生菌转录系统预计不像真核生物后代那样复杂；(C)鉴于转录复杂成分及其相互作用的高温来源，预计它们将更加强大。在使用RNAPII进行蛋白质编码基因的真核转录中，PIC的组装始于TATA结合蛋白(TBP)对转录起始点上游约25bp的含有TATA的启动子序列的识别。然后，转录因子(TF)IIB与TBP/启动子复合体结合，并将RNAPII和其他转录因子招募到复合体中启动转录。该提案侧重于TBP和TFIIB的古生代同源物。关于EucarYal TFIIB与TBP/启动子相互作用的结构数据可用；它们表明TFIIB约200个残基的C-末端核心与TBP结合，以及TATA盒上下游的DNA序列，后者与TBP相互作用。目前还没有关于真核生物TFIIB的N-端CA 100残基处置的结构信息。我们的工作阐明了P.Furiosus TFB(与eucarYal TFIIB同源)一个50个残基的N-末端片段的核磁共振溶液结构，它采用了与转录延伸因子TFIIS的C-末端结构域非常相似的“锌带”结构。TFIIB的C-末端部分的DNA结合能力表明，TFIIB的N-末端结构域可能与TATA-box下游的DNA相互作用，可能位于转录起始点附近。已知真核生物TFIIB N-末端结构域的点突变会影响转录起始点的位置。这种独特的结构信息将用于研究TFIIB的N-末端部分的结构、相互作用和功能。核磁共振将被用来表征P.Furiosus TFB的一个更大的(125个残基)N-末端片段的结构，为组装全长蛋白质的结构模型提供背景。核磁共振也将用于解决人TFIIB的N末端片段的溶液结构，以与P.Furiosus TFB的溶液结构进行比较。这一点很重要，因为与古生菌(和酵母)中的四种半胱氨酸相比，高等真核生物具有推测的(半胱氨酸、组氨酸、半胱氨酸、半胱氨酸)锌结合残基。建立了一种灵敏的方法，利用拴系铁(EDTA)作为局部人工核酸酶来探测与P.Furiosus TFB的N(和C-)末端区域接触的DNA区域。将利用已建立的来自嗜热甲烷球菌转录成分的杂交无细胞系统开发一种功能性转录测试。这一方法和核酸酶探针法将被用来研究P.Furiosus TFB的N-末端区域的靶向突变的影响。特别令人感兴趣的是锌结合对转录起始中TF(II)B正常功能的要求。最后，将使用几种策略来确定P.Furiosus转录装置的其他必需组件，包括RNA聚合酶。最终，一个在体外发挥作用的基础转录复合体将使对P.Furiosus转录调控的研究成为可能。由编码蛋白质的基因产生的蛋白质包括将DNA(基因)转录成信使RNA，然后将信使RNA翻译成蛋白质。例如，对参与转录和调控这一过程的蛋白质的详细描述有望为遗传病的治疗提供靶标。生命的古生物领域是在过去十年中发现的，它是一组独立的微生物，既不同于细菌，也不同于真核生物。古生菌的转录系统似乎与真核生物的转录系统关系更密切，而不是细菌的转录系统。该项目将研究古生菌转录复合体的拓扑结构(蛋白质和核酸成分的排列)，以发现：(A)古生菌复合体功能的更多细节，目前对此知之甚少；(B)古生菌转录复合体是否独特，能够产生所有三种类型的RNA(mRNA、rRNA、tRNA)；以及(C)超高温海洋古生型呋喃热球菌的转录系统如何在100摄氏度的最佳生长温度下发挥其功能。我们将在已有的关于真核转录复合体成分的结构工作的基础上，研究动脉成分。特别令人感兴趣的是转录因子IIB的N-末端区域与转录起始点附近的启动子DNA可能的相互作用，几组实验的目的是阐明这些相互作用。*？？

Scott 9631093 Biophysical and biochemical methods will be employed to study the structure and assembly of protein and DNA components of the transcription pre-initiation complex of the hyperthermophilic archaeon Pyrococcus furiosus. The single archaeal transcription system exhibits more homology to the eucaryal than the bacterial transcription apparatus, suggesting that an ancestral archaeal system evolved into the three eucaryal transcription complexes that synthesize rRNA, mRNA, and tRNA, using RNA polymerase (RNAP)I, II, and III, respectively. The resulting understanding of the molecular topology and mechanism of archaeal basal transcription is expected to shed light on eucaryal transcription as well as providing evolutionary information related to the divergence of the three eucaryal systems. The P. furiosus transcription system was chosen for study because: (a) little is known about archaeal transcription and its regulation; (b) the archaeal system is expected to be less complex than the eucaryal descendants; (c) the transcription complex components and their interactions are expected to be more robust given their hyperthermophilic source. In eucaryal basal transcription of protein-coding genes using RNAPII, the assembly of the PIC starts with recognition by the TATA-binding protein (TBP) of TATA-containing promoter sequences ca. 25 bp upstream of the transcription start site. Then, transcription factor (TF)IIB binds to the TBP/promoter complexes and recruits RNAPII and other transcription factors to the complex to initiate transcription. This proposal focuses on archaeal homologs of TBP and TFIIB. Structural data are available on the interaction of eucaryal TFIIB with TBP/promoter; they indicate that the ca. 200 residue C-terminal core of TFIIB binds to TBP and DNA sequences both up- and downstream of the TATA box, which interacts with TBP. No structural information is available on the disposition of the N-terminal ca 100 residues of eucaryal TFIIB . Our work has elucidated the NMR solut ion structure of a 50 residue N-terminal fragment of P. furiosus TFB (homologous to eucaryal TFIIB), which is shown to adopt a "zinc ribbon" structure very similar to that displayed by the C-terminal domain of human TFIIS, a transcription elongation factor. The DNA-binding ability of the C-terminal portion of TFIIB suggests that the N-terminal domain of TFIIB may interact with the DNA downstream of the TATA--box, perhaps near the transcription start site. Point mutations in the N-terminal domain of eucaryal TFIIB are known to affect the location of the transcription start site. This unique structural information will be put to use in studies designed to probe the structure, interactions, and function of the N-terminal portion of TFIIB. NMR will be used to characterize structurally a larger (125 residue) N-terminal fragment of P. furiosus TFB, to provide the context to assemble a structural model for the full-length protein. NMR will also be used to solve the solution structure of the N-terminal fragment of human TFIIB for comparison with that of P. furiosus TFB. This is important since higher eucarya have putative (Cys, His, Cys, Cys) zinc binding residues compared to four Cys in archaea (and yeast). A sensitive method will be developed using tethered Fe(EDTA) as a local artificial nuclease to probe the region of DNA contacted by the N(and C-)terminal regions of P. furiosus TFB. A functional transcription assay will be developed using an established hybrid cell-free system from Methanococcus thermolithotrophicus transcription components. This and the nuclease-probe method will be used to study the effects of targeted mutations in the N-terminal domain of P. furiosus TFB. Of particular interest is the requirement of zinc binding for proper functioning of TF(II)B in transcription initiation. Finally, several strategies will be used to identify other required components of the P. furiosus transcription apparatus, including the RNA polymerase. Ultimately, a functioning in vitro basal transcription complex will allow the study of transcriptional regulation in P. furiosus. %%% Production of proteins from the genes that encode them consists of transcription of the DNA (gene) to messenger RNA, then translation of the mRNA into protein. A detailed picture of the proteins involved in transcription and the regulation of this process is expected to provide, for example, targets for therapy of genetic diseases. The archaeal domain of life was discovered in the past decade as a separate group of microorganisms distinct from bacteria as well as from eukaryotes. The transcription system of archaea appears to be more closely related to that of eukaryotes, rather than that of bacteria. This project will study the topology (arrangement of protein and nucleic acid components ) of the archaeal transcription complex to discover: (a) more details of the functioning of the archaeal complex, about which little is known; (b) whether the archaeal transcription complex is unique in being able to produce all three types of RNA, (mRNA, rRNA, tRNA); and (c) how the transcription system of he hyperthermophilic marine archaeal Pyrococcus furiosus can carry out its function at the optimal growth temperature of 100 degreeC. We will build on structural work already available on components of the eukaryotic transcription complex to study the arterial components. Of particular interest is the possible interaction of the N-terminal domain of transcription factor IIB with promoter DNA near the transcription start site and several sets of experiments are targeted at elucidating these interactions. *** ??