比較原核及低等真核細胞(酵母菌)的tRNA synthetase可以發現：許多酵母菌的細胞質tRNA synthetase在其N或C端會多出一段多胜肽，稱為附屬區段，這些附屬區段不存在原核細胞的相對應酵素，而且在其序列中常包含許多帶正電荷的lysine胺基酸，因此能非專一性地結合帶負電荷的化合物(如RNA)。附屬區段除了會結合RNA外，也有少部分的附屬區段參與了蛋白質-蛋白質之間的交互作用，例如glutamyl-tRNA synthetase及methionyl-tRNA synthetase便是其中較出名的例子。最近的研究更發現不少酵母菌tRNA synthetase的附屬區段含有細胞核標的訊號(NLS)，這些酵素可能藉由這些獨特的標的訊號進入細胞核內從事一些非傳統的功能(即胺醯化以外的功能)。但是總體而言，附屬區段對tRNA synthetase的生物功能或生化活性有何重要性還是不清楚。序列比對的結果顯示酵母菌valyl-tRNA synthetase (ValRS)的N端也有一段帶正電荷的附屬區段，長度約含97個胺基酸，這個序列並未出現在大腸桿菌的同源酵素內。有趣的是：人類ValRS 的N端除了有類似的帶正電荷附屬區段 (~胺基酸 201-300)外，它還多了一段厭水性的附屬區段(~胺基酸 1-200)，這個厭水性的區段已經被証實會與轉譯延長因子 EF-1H作用，這個交互作用一方面可以促進ValRS的胺醯化效率.，另一方面可以將valyl-tRNA由ValRS直接轉運到核醣體。截至目前為止，酵母菌ValRS附屬區段或人類ValRS相對應序列(~胺基酸 201-300)的真正功能尚不清楚。本計畫為期三年，在此期限內我們將針對此附屬區段作深入的研究及探討。重點包括： (1) 鑑定及分析以下數個蛋白質的tRNA結合親和力及專一性：附屬區段、野生株ValRS、刪除附屬區段的ValRS; (2) 比較野生株ValRS及刪除附屬區段的ValRS之細胞內功能、胺醯化活性、及酵素動力學數值 (KM及 kcat); (3)利用Yeast Two-Hybrid系統及免疫沉澱法篩選出會與附屬區段有專一性交互作用的蛋白質，進而研究它可能衍生出的非傳統功能; (4)鑑定S. pombe的真正ValRS基因。 Many yeast cytoplasmic tRNA synthetases contain an amino- or carboxyl-terminal polypeptide extension which is absent from their bacterial homologues. These extensions are generally rich in lysine residues and capable of binding non-specifically to polyanionic carriers such as RNA. In addition, some appended domains of yeast tRNA synthetases are found to participate in protein-protein interactions, such as those of yeast glutamyl- and methionyl-tRNA synthetases (GluRS and MetRS). Furthermore, many of these appended domains contain one or several classical nuclear localization signals (NLSs) which are thought to play a role in nuclear import of these otherwise “cytoplasmic” proteins. However, more generally, the exact role of the appended domains in the biological functions of these tRNA-charging enzymes remains elusive. Sequence comparison suggests that yeast valyl-tRNA synthetase (ValRS) also contains a lysine-rich polypeptide extension of 97 residues. In contrast to the yeast enzyme, human ValRS has conserved the positively charged N-terminal extension (~ residues 201-300) that distinguishes the yeast enzyme from its bacterial counterparts, while acquiring an additional hydrophobic domain (~ residues 1-200) that is responsible for its interaction with the elongation factor EF-1H. Such an interaction was shown to be essential for stimulation of its aminoacylation activity and direct transfer of valyl-tRNA from the synthetase to ribosome. So far, little is know about the biological function of the Ad of yeast ValRS or its equivalent sequence in the human enzyme (~ residues 201-300). The present proposal is a 3-year project in which we wish to investigate the functional relevance of the appended domains of yeast and human ValRSs to their respective enzymes and further characterize their biochemical activities; the main points include: (1) elucidating the tRNA-binding affinities and specificities of the appended domain, full-length ValRS, and N-terminally truncated ValRSs in vitro; (2) determining the aminoacylation activities and kinetic parameters for tRNA(Val) of the full-length and truncated ValRSs; (3) identifying the interacting partners of yeast ValRS by yeast two-hybrid screening and co-immunoprecipitation; (4) identifying the authentic ValRS gene of S. pombe. 研究期間：9908 ~ 10007
