臺灣博碩士論文加值系統

tRNAHis上額外的5'鳥嘌呤核苷酸（G-1）是這種tRNA在大多數生物中普遍的特徵，同時也是tRNAHis上的主要辨識元素(Identity element)。G-1來自於基因編碼或通過tRNAHis鳥苷酸轉移酶（tRNAHis guanylyltransferase，簡稱Thg1）轉錄後修飾添加。儘管秀麗隱桿線蟲(Caenorhabditis elegans)是一種不具有Thg1的生物，但它的細胞質tRNAHis基因保留了G-1的編碼。我們的研究表明，其線粒體tRNAHis缺乏G-1。這種tRNA雖然缺乏典型的辨識元素G-1，但仍可在體內有效地被氨酰基化。點突變實驗結果顯示，反密碼子對線蟲tRNAHis的識別效率影響很大，幾乎與典型的辨識元素G-1相當。另一方面，N73的鹼基也是tRNAHis的關鍵辨識元素。人類基因體中擁有兩種截然不同的組氨酰-tRNA合成酶（histidyl-tRNA synthetase，簡稱HisRS）同源基因，儘管這兩種酵素具有高度序列相似性（81％相似性），但它們強烈偏好不同的辨識元素（A73或C73）。最有趣的是，在細胞質和線粒體tRNAHis之間交換辨識元素（A73和C73）可以改變人類HisRS對這兩種tRNA的專一性。此外，人類只擁有單個Thg1基因，但同時負責修飾細胞質（含A73）和線粒體（含C73）tRNAHis。我們實驗結果顯示人類細胞質tRNAHis是通過ATP依賴機制被修飾，而其線粒體tRNAHis通過GTP依賴機制被修飾。這兩種機制都僅將一個G殘基加在tRNAsHis上。雖然N73影響Thg1添加G-1的效率，但N73的突變（A73至C或C73至A）並不改變修飾機制的種類。最有趣的是，GTP依賴機制會被ATP抑制。這篇論文中我們不僅發現了線蟲獨特的tRNAHis辨識機制，還分析了人類兩個同源HisRS酵素專一性的分化以及兩種獨特地tRNAHis的轉錄後修飾的機制。

The extra 5’ guanine nucleotide (G-1) on tRNAHis is a nearly universal feature that specifies tRNAHis identity. The G-1 residue is either genome encoded or post-transcriptionally added by tRNAHis guanylyltransferase (Thg1). Despite Caenorhabditis elegans being a Thg1-independent organism, its cytoplasmic tRNAHis retains a genome-encoded G-1. Our study showed that its mitochondrial tRNAHis lacks G-1. This tRNA, while lacking the canonical identity element, can still be efficiently aminoacylated in vivo. Mutagenesis assays showed that the anticodon takes a primary role in C. elegans tRNAHis identity recognition, being comparable to the universal identity element. On the other hand, the discriminator base N73 is a key identity element of tRNAHis. Humans possess two distinct yet closely related histidyl-tRNA synthetase (HisRS) homologues, despite these two isoforms sharing high sequence similarities (81% identity), they strongly preferred different discriminator bases (A73 or C73). Most intriguingly, swapping the discriminator base between the cytoplasmic and mitochondrial tRNAHis isoacceptors conveniently switched their enzyme preferences. Moreover, humans possess a single Thg1 gene is responsible for modification of both the cytoplasmic (with A73) and mitochondrial (with C73) tRNAHis isoacceptors. We reported herein that human cytoplasmic tRNAHis was modified via an ATP-dependent mechanism, while its mitochondrial tRNA isoacceptor was modified via a GTP-dependent mechanism. While N73 affected the efficiency of G-1 addition by Thg1, mutation of N73 (A73 to C or C73 to A) did not switch the mechanisms used for modification. Most intriguingly, the GTP-dependent mechanism was inhibited by ATP. Our study reveals novel scenarios of recognition and modification of tRNAHis in higher eukaryotes. 

中文摘要 i
Abstract ii
誌謝 iii
List of Figures v
List of Tables vi
Overall introduction 1
Chapter I - Naturally occurring dual recognition of tRNAHis substrates with and without a universal identity element 5
Abstract 6
Materials and Methods 7
Results 12
Discussion 21
Chapter II - Evolutionary gain of highly divergent tRNA specificities by two isoforms of human histidyl-tRNA synthetase 24
Abstract 25
Materials and Methods 26
Results 28
Discussion 37
Chapter III - Human Thg1 adds G-1 to tRNAsHis through two alternative mechanisms 40
Abstract 41
Materials and Methods 42
Results 44
Summary 50
References 51
Figures and Tables 56
Appendix 78

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