Eukaryotic tRNA nucleotidyltransferase: synthesis, sorting and mechanism

转移RNA（tRNA）是衔接分子，在信息从DNA（其存储形式）到蛋白质（其功能形式）的转换中发挥重要作用。在像我们这样的生物体中，如果没有tRNA核苷酸转移酶的帮助，tRNA就不能发挥作用。这种酶在细胞中需要tRNA的每个位置（细胞质、线粒体、质体）都发挥作用。如果我们缺乏tRNA核苷酸转移酶，我们就缺乏功能性的tRNA，我们就不能制造蛋白质，我们就会死亡。鉴于这种酶的重要性，我们必须回答一些重要的问题。例如，细胞核中的单个基因如何编码细胞质、线粒体和质体中所需的多种形式的这种酶？酶上有什么信号告诉细胞将其发送到哪里，细胞中有什么因子识别这些信号？为了回答这些问题，我们将使用诱变来改变酶的形式（具有或缺乏潜在的靶向信号），并通过将它们连接到易于检测的报告蛋白来跟踪它们在细胞中的位置。酵母和植物的tRNA核苷酸转移酶比细菌的大得多（至少大三分之一）。coli）等。为什么它们更大，我们能将抗真菌剂或除草剂靶向这些额外的区域吗？为了探索这一点，我们正在对较大的tRNA核苷酸转移酶的独特区域进行改变，以观察这些改变的酶是否仍然支持酵母的生命，或者当纯化时，在试管中具有催化活性。我们将使用酶和生物物理技术，看看这些变化如何改变酶的结构和功能。最后，我们制造了这种酶的突变体形式，它对升高的温度非常敏感，我们发现了第二个位点的“抑制”突变，可以恢复活性。突变残基之间的相互作用可以告诉我们关于这种重要酶的结构和功能以及温度如何影响它的信息？

Transfer RNAs (tRNAs) are adaptor molecules that play an essential role in the conversion of information from DNA (its storage form) to protein (its functioning form). In organisms like us tRNAs do not work without the help of the enzyme tRNA nucleotidyltransferase which assists in their production. This enzyme functions in every location (cytosol, mitochondria, plastids) in a cell where tRNAs are needed. If we lack tRNA nucleotidyltransferase, we lack functional tRNAs, we can not make proteins and we die. Given the importance of this enzyme, we must answer some important questions. For example, how does a single gene residing in the nucleus code for multiple forms of this enzyme required in the cytosol, mitochondria and plastids? What are the signals on the enzyme that tell the cell where to send it and what factors in the cell recognize these signals? To answer these questions we will use mutagenesis to make altered forms of the enzyme (having or lacking potential targeting signals) and follow where they go in cells by linking them to easily detectable reporter proteins. The yeast and plant tRNA nucleotidyltransferases are much larger (at least one-third bigger) than their bacterial (E. coli) equivalent. Why are they bigger and can we target antifungal agents or herbicides to these extra regions? To explore this we are making changes in regions unique to the larger tRNA nucleotidyltransferases to see if these altered enzymes still support the life of yeast or, when purified, have catalytic activity in a test tube. We will use enzymatic and biophysical techniques to see how these changes alter enzyme structure and function. Finally, we made a mutant form of this enzyme that is extremely sensitive to elevated temperature and we found second site "suppressor" mutations that restore activity. What can the interactions between the mutant residues tell us about the structure and function of this important enzyme and how temperature affects it?