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Structural studies of prokaryotic and eukaryotic oligoribonucleases.

机译：原核和真核寡核糖核酸酶的结构研究。

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摘要

RNA metabolism includes all the processes required for RNA synthesis, maturation, and degradation in living cells. Ribonucleases (RNases) are involved in RNA maturation and degradation, two essential processes in gene expression and regulation in both prokaryotes and eukaryotes. Oligoribonuclease (Orn) has an important role in eliminating small oligonucleotides (nano-RNA), the last step in mRNA degradation.;In E. coli, Orn is the only essential exoribonuclease. The enzyme has been shown to form a stable dimer, both in solution and in the crystalline form. Analysis of the three-dimensional structure of Orn allowed us to hypothesize that dimerization is essential for enzyme catalysis. In order to test the hypothesis, I analyzed a number of deletion and point mutants of Orn and determined that tryptophan 143 is essential for dimerization. A W143A mutant is unable to dimerize and has very little activity, similar to that of an active site mutant (D162A). The atomic structure of the W143A mutant, solved at a resolution of 1.9 A, showed that although the overall three-dimensional fold is similar to that of the wild-type protein, minor differences exist that could account for the monomeric behavior in solution. A flexible Arg174 is repositioned into the cavity created by the missing Trp143. In this new orientation Arg174 protrudes into a hydrophobic pocket in the dimerization interface and is proposed to produce sufficient unfavorable interactions to keep the monomers apart in solution. All these data suggest that dimerization of Orn is essential for its activity.;The human homolog of Orn, also known as small fragment nuclease (Sfn), has been shown to degrade short single-stranded RNA, the last step in mRNA decay. In order to determine the mechanism of action of Sfn and its role in the cell, we solved the crystal structure of a truncated form of Sfn at a resolution of 2.6 A. This mutant form of Sfn lacks the C-terminal 21 amino acids (Sfn-DeltaC21) yet is as efficient as full length Sfn on model substrates. Interestingly, Sfn is not as active as E. coli Orn in in vitro assays. Analysis of the atomic structure revealed that the active site cleft in Sfn is narrower than the corresponding active site in E. coli. We propose a model for how this narrower cleft may explain the lower in vitro activity.;Bacillus subtilis does not have an Orn homolog and until recently, the enzyme responsible for nano-RNA degradation in this organism was unknown. Ytql (also termed nrnA or nanoRNase), a protein unrelated to E. coli Orn, was recently shown to be responsible for the digestion of oligonucleotides in B. subtilis. In order to better understand the mechanism of action of Ytql, I solved its crystal structure at a resolution of 2.0 A. The nuclease has a RecJ-like fold with two globular domains connected via a flexible linker that forms a central groove. On one side of the groove, the larger N-terminal domain harbors the putative active site, while on the opposite side, the C-terminal domain includes a putative RNA binding domain. The structure of Ytql provides insights into how this enzyme binds and digests oligoribonucleotides.;The studies described here provide a better understanding of the mechanism of action for several exoribonucleases that act on nano-RNA oligonucleotides---Oligoribonuclease from E. coli, its close homolog in humans (Small fragment nuclease), as well as a functional homolog in Bacillus (Ytql). This work is relevant to understanding RNA metabolism, which is an essential process for survival of both eukaryotic and prokaryotic organisms.
机译：RNA代谢包括活细胞中RNA合成，成熟和降解所需的所有过程。核糖核酸酶（RNA酶）参与RNA的成熟和降解，这是原核生物和真核生物在基因表达和调控中的两个基本过程。寡核糖核酸酶（Orn）在消除小寡核苷酸（纳米RNA）（mRNA降解的最后一步）中起着重要作用。在大肠杆菌中，Orn是唯一必需的核糖核酸外切核酸酶。已显示该酶在溶液和晶体形式下均能形成稳定的二聚体。对Orn三维结构的分析使我们假设二聚化对于酶催化至关重要。为了检验该假设，我分析了Orn的许多缺失和点突变，并确定色氨酸143对于二聚化至关重要。与活性位点突变体（D162A）相似，W143A突变体无法二聚化并且几乎没有活性。 W143A突变体的原子结构以1.9 A的分辨率解析，表明尽管整体三维折叠与野生型蛋白质相似，但仍存在细微差异，这可以解释溶液中的单体行为。将一个灵活的Arg174重新放置到缺少的Trp143创建的空腔中。在这个新的方向上，Arg174突入二聚界面中的疏水袋中，并被提议产生足够不利的相互作用，使单体在溶液中保持分开。所有这些数据表明Orn的二聚化对其活性至关重要。Orn的人类同源物，也称为小片段核酸酶（Sfn），已显示可降解短的单链RNA，这是mRNA降解的最后一步。为了确定Sfn的作用机理及其在细胞中的作用，我们以2.6 A的分辨率解析了Sfn的截短形式的晶体结构。Sfn的这种突变形式缺少C端21个氨基酸（Sfn -DeltaC21）在模型基板上的效率与全长Sfn一样。有趣的是，在体外测定中，Sfn的活性不如大肠杆菌Orn。原子结构分析表明，Sfn中的活性位点裂隙比大肠杆菌中相应的活性位点窄。我们提出了一个模型，用于解释这种狭窄的裂口如何解释较低的体外活性。枯草芽孢杆菌没有Orn同源物，直到最近，这种生物中负责纳米RNA降解的酶还是未知的。 Ytql（也称为nrnA或nanoRNase），一种与大肠杆菌Orn不相关的蛋白质，最近被证明负责枯草芽孢杆菌中寡核苷酸的消化。为了更好地理解Ytql的作用机理，我以2.0 A的分辨率解析了它的晶体结构。核酸酶具有类似RecJ的折叠结构，其中两个球状结构域通过形成中心凹槽的柔性接头连接。在凹槽的一侧，较大的N末端结构域带有推定的活性位点，而在另一侧，C末端结构域包含推定的RNA结合结构域。 Ytql的结构提供了对该酶如何结合和消化寡核糖核苷酸的见解。这里描述的研究提供了对几种作用于纳米RNA寡核苷酸的外切核糖核酸酶的作用机理的更好理解-来自大肠杆菌的寡聚核糖核酸酶，人类的同系物（小片段核酸酶），以及芽孢杆菌的功能同系物（Ytql）。这项工作与了解RNA代谢有关，RNA代谢是真核生物和原核生物存活的重要过程。

著录项

  • 作者

    Nelersa, Claudiu Marius.;

  • 作者单位

    University of Miami.;

  • 授予单位 University of Miami.;
  • 学科 Biology Molecular.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 104 p.
  • 总页数 104
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;生物化学;
  • 关键词

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