Topology of Archaeal Transcription Pre-Initiation Complex

Scott 9631093采用生物物理和生物化学方法研究极端嗜热古菌Pyrococcus furiosus转录前起始复合物的蛋白质和DNA组分的结构和组装。单一的古细菌转录系统表现出更多的同源性，真核比细菌的转录装置，这表明一个古老的古细菌系统进化成三个真核转录复合物，合成rRNA，mRNA和tRNA，使用RNA聚合酶（RNAP）I，II和III，分别。由此产生的古细菌基础转录的分子拓扑结构和机制的理解，有望揭示真核转录，以及提供相关的进化信息的分歧的三个真核系统。选择P. furiosus转录系统进行研究是因为：（a）对古细菌转录及其调控知之甚少;（B）古细菌系统预计比真核后代复杂性低;（c）鉴于其超嗜热来源，转录复合物组分及其相互作用预计更稳健。 在使用RNAPII的蛋白质编码基因的真核基础转录中，PIC的组装始于TATA结合蛋白（TBP）对含有TATA的启动子序列ca.转录起始位点上游25 bp处。然后，转录因子（TF）IIB结合TBP/启动子复合物，并将RNAPII和其他转录因子募集到复合物中以启动转录。该提案侧重于TBP和TFIIB的古细菌同系物。关于真核TFIIB与TBP/启动子的相互作用的结构数据是可用的;它们表明ca. TFIIB的200个残基C-末端核心结合TBP和TATA盒上游和下游的DNA序列，TATA盒与TBP相互作用。没有关于真核TFIIB的N-末端约100个残基的处置的结构信息。我们的工作已经阐明了一个50个残基的N-末端片段的NMR溶液结构的P. furiosus TFB（同源于真核TFIIB），它被证明采用一个“锌带”结构非常类似的C-末端结构域的人TFIIS，转录延伸因子。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-盒下游的DNA相互作用，可能在转录起始位点附近。已知真核TFIIB的N-末端结构域中的点突变影响转录起始位点的位置。 这种独特的结构信息将被用于设计用于探测TFIIB的N-末端部分的结构、相互作用和功能的研究。NMR将用于在结构上表征激烈变形杆菌TFB的较大（125个残基）N-末端片段，以提供组装全长蛋白质的结构模型的背景。NMR还将用于解析人TFIIB的N-末端片段的溶液结构，以与激烈变形杆菌TFB的溶液结构进行比较。这是重要的，因为与古细菌（和酵母）中的四个Cys相比，高等真核生物具有推定的（Cys、His、Cys、Cys）锌结合残基。将开发一种灵敏的方法，使用拴系的Fe（EDTA）作为局部人工核酸酶，以探测与P. furiosus TFB的N（和C-）末端区域接触的DNA区域。一个功能性的转录测定将开发使用一个已建立的杂交无细胞系统从嗜热嗜石甲烷球菌转录组件。该方法和核酸酶探针方法将用于研究在P. furiosus TFB的N-末端结构域中的靶向突变的影响。特别令人感兴趣的是在转录起始中TF（II）B的适当功能需要锌结合。 最后，将使用几种策略来鉴定激烈毕赤酵母转录装置的其他必需组分，包括RNA聚合酶。最终，一个功能性的体外基础转录复合物将允许在P. furiosus中研究转录调控。 从编码蛋白质的基因中产生蛋白质，包括DNA（基因）转录成信使RNA，然后将mRNA翻译成蛋白质。 参与转录和调节这一过程的蛋白质的详细图片预计将提供，例如，遗传疾病的治疗目标。 在过去的十年中，古细菌的生命领域被发现是一组独立的微生物，与细菌和真核生物不同。古生菌的转录系统似乎与真核生物的转录系统更密切相关，而不是细菌的。 本项目将研究（蛋白质和核酸组分的排列）以发现：（a）关于古细菌复合体的功能的更多细节，对此知之甚少;（B）古细菌转录复合体是否在能够产生所有三种类型的RNA方面是独特的，（mRNA、rRNA、tRNA）;以及（c）极端嗜热海洋古细菌激烈火球菌（Pyrococcus furiosus）的转录系统如何在100 ℃的最佳生长温度下发挥其功能。 我们将建立在结构上的工作已经对真核转录复合物的组成部分，研究动脉成分。 特别感兴趣的是可能的相互作用的N-末端结构域的转录因子IIB与启动子DNA附近的转录起始位点和几组实验的目标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. *** ??