Investigation on the Read-through Phenomenon of Pre-mature Stop Codons

• Other Authors: Wong Wai Hung (author.)
• Format: Others
• Language: English; Chinese
• Published: 2017
• Online Access: http://repository.lib.cuhk.edu.hk/en/item/cuhk-1292327

有一件對於研究生物學的學術人士最正常的事情是當核糖體負責翻譯識別任何三種終止密碼子，一個釋放因子結合核酶和釋放的同時結束翻譯的增長的肽鏈上。但是最令人驚奇的是居然會有實驗發現，這種終止密碼子的終止進程也有可能會被提前抑制，而且被翻譯成氨基酸。在另一方面，無意義抑制傳送核糖核酸被發現及被證明能夠蛋白翻譯過程中把特殊氨基酸摻入正在增長的肽鏈上。第21 種氨基酸，硒半胱胺酸，是這些能被摻入的修飾氨基酸中的其中一個，它是專門通過無意義抑制傳送核糖核酸識別UGA 終止密碼子進行。除了這個發現外，第22種氨基酸吡咯賴胺酸，也已被證明能利用無意義抑制傳送核糖核酸和傳送核糖核酸合成酶來直接摻入正在增長的肽鏈上的。在這裡，我提出一頂名為蛋白質翻譯修改新機制，在這當中預先已被共價修飾的氨基酸會被直接在翻譯過程中納入肽鏈。另外，作為組蛋白這樣一個如此高程度修飾的蛋白質，能有其中一種組蛋白修飾的可能性，是以這裹所提出的特殊機制去實現？所以為了驗證這一個假設，重組熒光素酶蛋白的質體被選為報告的載體。然後，執行熒光素酶活性測定和蛋白質印跡以證明經轉染的C2C12 小鼠成肌細胞系能在體外展示讀通現象。為了更深層次的去探討讀通現象，我們已經拜託了劍橋大學的一個研究小組分享一個傳送核糖核酸合成酶的質體和自行購買了一個立即可用的已修飾氨基酸，乙酰基賴氨酸。這兩種元素能分別在轉染的階段被加入，看看它們分別在讀通現象中能獨立呈現的效果。這項研究的結論是，在C2C12 這已經證明讀通現象的細胞系中，加入乙酰賴氨酸可以在UGA 和UAA 終止密碼子中上調讀通現象，不過加入傳送核糖核酸合成酶只能在UGA 終止密碼子中上調通讀現象密碼而已。 === It is the most sensible thing for everybody studying Biology – when ribosome which is responsible for translation recognizes any of the three stop codons, a release factor binds to the ribozyme and releases the growing peptide chain ending translation at the same time. It is surprising to all scientists to find out that this termination progress could possibly be suppressed pre-maturely and translated into amino acids. On the other hand, non-sense suppressor tRNAs have been discovered and proven to be capable to incorporate special amino acids into the growing peptide chains during protein translation. The 21st amino acid, selenocysteine, is one of these incorporating modified amino acids and it is specifically carried by non-sense suppressor tRNA recognizing UGA stop codon. Apart from that, direct incorporation of pyrrolysine, the 22nd amino acid, was demonstrated making use of non-sense suppressor tRNA and tRNA synthetase. Here, I am proposing a novel mechanism called Protein Translating Modification, in which previously modified amino acids are incorporated into peptide chains directly during translation. In addition, as histone protein is such a heavily modified protein (predominantly at its N-terminal regions), is there a possibility for histone modifications of such “post-translational modifications” to be achieved by this mechanism? So in order to test the hypothesis, a recombinant luciferase-histone plasmid is constructed as reporter vector. Then, luciferase activity assay and Western Blotting will be performed to demonstrate the in vitro read-through phenomenon in the transfected C2C12 mouse myoblast cell lines. To look into the details of the read-through phenomenon, a tRNA amino-acyl synthetase bearing plasmid has been obtained from a research team (Professor Jason Chin) of the University of Cambridge and the immediately available modified (labelled) amino acids, acetyl-lysine, have also been purchased and/or synthesized. These two components could be added separately during transfection stage to see what are their separate effects contributing to the read-through phenomenon. The conclusion for this study is that in C2C12 this already demonstrating read-through phenomenon cell line, the addition of acetyl-lysine could up-regulate the read-through phenomenon in ochre and amber stop codons while the addition of the tRNA synthetase could only up-regulate the read-through phenomenon in amber stop codon only. === Wong Wai Hung. === Thesis M.Phil. Chinese University of Hong Kong 2017. === Includes bibliographical references (leaves ). === Abstracts also in Chinese. === Title from PDF title page (viewed on …). === Detailed summary in vernacular field only.