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研究生:張勵仁
研究生(外文):Chang, Li-Ren
論文名稱:大腸桿菌之核醣核酸降解體在5S核醣核酸的成熟上所扮演的角色
論文名稱(外文):Role of the RNA degradosome in the processing of 5S rRNA in escherichia coli: The RNA degradosome is also a processosome
指導教授:林淑端林淑端引用關係
指導教授(外文):Lin, Sue-Chao
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:英文
中文關鍵詞:核醣核酸內切脢E5S核醣核酸生物化學生物學科學核醣核酸降解體蛋白質複合體
外文關鍵詞:RNase E5S rRNABIOCHEMISTRYBIOLOGYSCIENCERNA degradosome
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核醣核酸內切酉每E是大腸桿菌之一個必須酵素,它在訊息核醣核酸的穩定性及核糖體核醣核酸的成熟上扮有關鍵性的角色。早期的研究發現在5S核醣核酸的成熟,必須先由核醣核酸內切酉每E切割其前驅物9S核醣核酸,才能進一步由核醣核酸外切酉每將其修飾成熟。近年來核醣核酸內切酉每E已被證明能夠以蛋白質複合體的形式存在,被稱之為『核醣核酸降解體』。在此複合體中,到目前為止已發現了六種蛋白質:除了核醣核酸內切酉每E之外,尚包括核醣核酸外切酉每PNPase、核醣核酸螺旋醇(RhlB RNA helicase)、熱休克蛋白DnaK、糖解作用的烯醇酉每(enolase)及聚磷酸激酉每(polyphosphate kinase)。另外,複合體中也含有不同種類之核醣核酸成員,如訊息核醣核酸、16S及23S核糖體核醣核酸降解的產物。目前証據都顯示此核醣核酸降體主要為執行核醣核酸降解。然而,5S核醣核酸是否在核醣核酸降解體中成熟仍然是個謎。
經由大量表現5S核醣核酸的前驅物,由北方轉漬法分析中發現了突變核醣核酸內切酉每E所分離的核醣核酸降解體中有5S核醣核酸的前驅物。進一步利用細胞內放射性標記核醣核酸的方法,更可以觀察到5S核醣核酸的前驅物在核醣核酸降解體中的變化。分析比較在細胞內全體及核醣核酸降解體內的5S核醣 核酸,發現 5S核醣核酸在核醣核酸降解體中是已經完全成熟的。由於5S核醣核酸的成熟也需要核醣核酸外切酉每T的作用,而之前的研究並未發現核醣核酸降解體內有核醣核酸外切酉每T 的存在;核醣核酸外切酉每T是否為一個尚未被發現的成員,軌 成了一個重要的問題。利用西方轉漬法分析核醣核酸降解體, 果然發現核醣核酸外切酉每T存在於此複合體中。
此篇研究報告證明5S核醣核酸的成熟可在核醣核酸降解體中進行。因此,本研究結果顯示此核醣核酸內切酉每E所形成的蛋白質複合體不但是一個『核醣核酸降解體』,也是一個『加工成熟體』。
RNase E, an essential endoribonuclease in E. coli, plays an important role in maturation of 5S rRNA and degradation of many species of mRNA. Recently, RNase E has been shown to form a multicomponent ribonucleolytic complex termed RNA degradosome, in which, RNase E was found to be associated with PNPase, Rh1B RNA helicase, DnaK, enolase, polyphosphate kinase and RNA components. The major RNA species in the RNA degradosome are 23S and 16S rRNA-degrading products. However, the 5S rRNA together with some known RNase E substrates have also been detected in the degradosome.
It is known that, to produce a mature 5S rRNA, RNase E cleaves the precursor of 5S rRNA(9S RNA) in two sites and forms a premature product, which is subsequently processed by the RNase T (a 3' to 5' exoribonuclease) and unknown 5'-end-processing-enzyme. So far, there is no evidence showing that either RNase T is one of the components in RNA degradosome or the maturation of 5S rRNA indeed can occur in it. Thus, whether the RNA degradosome is also a processosome is still a mystery.
To answer whether the maturation of 5S rRNA can occur in the RNA degradosome, Northern blot analysis has been performed to show that both mature 5S rRNA and its precursor 9S RNA can exist in the degradosome. Subsequently, pulse labeling of 5S rRNA precursor using phosphorus-32 and followed by chase with cold phosphate have been done to study the kinetics of the processing of 5S rRNA in the degradosome. In this experiment, a heterogeneous species of 5S rRNA was detected. Using primer extension and RNA protection assay to analyze these 5S rRNAs from the RNA degradosome, it has been shown that both 5' and 3' regions of 5S rRNA are completely processed. These results suggest a possibility that both 3'-end-processing-enzyme (RNase T) and the unknown 5'-end-processing-enzyme can be associated with the complex. Interestingly, using anti-RNase T polyclonal antibodies and Western blot analysis, RNase T was detected in the RNA degradosome. In conclusion, it has been demonstrated that the RNA degradosome, in addition to degrading mRNAs and rRNAs, can also mature 5S rRNAs. Thus the RNA degradosome is also a processosome.

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