Studies of Trans-Acting TW17 ribozyme Catalysis Systems to Decipher Its Phosphorothiolate Thiolester Hydrolysis Mechanism
碩士 === 高雄醫學大學 === 醫藥暨應用化學研究所 === 99 === 英文摘要 The TW17 RNA is a unique Zn2+-dependent ribozyme capable of performing cis-acting catalysis to hydrolyze a phosphorothiolate thiolester bond between the RNA molecule and its substrate. The objectives of the study are to further decipher the reaction mecha...
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ndltd-TW-099KMC055370252015-10-13T20:37:29Z http://ndltd.ncl.edu.tw/handle/54991295890093656380 Studies of Trans-Acting TW17 ribozyme Catalysis Systems to Decipher Its Phosphorothiolate Thiolester Hydrolysis Mechanism 經由TW17核酸酶in trans 催化系統設計之應用於該磷酸硫酯鍵水解反應機制探討 Yi-Ming Liou 劉宜明 碩士 高雄醫學大學 醫藥暨應用化學研究所 99 英文摘要 The TW17 RNA is a unique Zn2+-dependent ribozyme capable of performing cis-acting catalysis to hydrolyze a phosphorothiolate thiolester bond between the RNA molecule and its substrate. The objectives of the study are to further decipher the reaction mechanism of TW17 ribozyme catalysis and improve its catalytic efficiency. We previously dissected the ribozyme into a trans-acting ribozyme system containing two RNA fragments: a smaller substrate-bearing RNA (18-mer) and a larger RNA containing the catalytic core (69-mer), and demonstrated catalytic efficiency of the trans-acting ribozyme system. We first purified the reaction product of the trans-acting TW17 ribozyme system and analyzed the acquired RNA by MALDI-TOF MS to unravel the substrate specificity of the ribozyme. The MALDI-TOF analysis intends to provide evidence that the reaction product is a GMP-primed 18-mer RNA and support the TW17 ribozyme as a phosphorothiolate thiolesterase RNA. We also reasoned that further enhancing adsorption of the substrate-bearing RNA on the catalytic RNA moiety could improve catalytic efficiency of the original trans-acting TW17 ribozyme system. Our studies indicated that the original trans-acting TW17 ribozyme system had satisfactory binding affinity with Kd of 0.18 ?b 0.06 ?嵱. When strengthening the binding stem between the two RNA moieties or extending their binding stem length, these structural-modified trans-acting TW17 ribozyme systems showed similar or inferior binding affinity (similar or higher Kd values) to that of the original one. Adsorption of the substrate RNA on the catalytic core RNA is thus unlikely the rate-determining step during catalysis. This study sheds light on the mechanism of TW17 ribozyme catalysis. Tzu-Pin Wang 王子斌 2011 學位論文 ; thesis 73 zh-TW |
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碩士 === 高雄醫學大學 === 醫藥暨應用化學研究所 === 99 === 英文摘要
The TW17 RNA is a unique Zn2+-dependent ribozyme capable of performing cis-acting catalysis to hydrolyze a phosphorothiolate thiolester bond between the RNA molecule and its substrate. The objectives of the study are to further decipher the reaction mechanism of TW17 ribozyme catalysis and improve its catalytic efficiency. We previously dissected the ribozyme into a trans-acting ribozyme system containing two RNA fragments: a smaller substrate-bearing RNA (18-mer) and a larger RNA containing the catalytic core (69-mer), and demonstrated catalytic efficiency of the trans-acting ribozyme system. We first purified the reaction product of the trans-acting TW17 ribozyme system and analyzed the acquired RNA by MALDI-TOF MS to unravel the substrate specificity of the ribozyme. The MALDI-TOF analysis intends to provide evidence that the reaction product is a GMP-primed 18-mer RNA and support the TW17 ribozyme as a phosphorothiolate thiolesterase RNA. We also reasoned that further enhancing adsorption of the substrate-bearing RNA on the catalytic RNA moiety could improve catalytic efficiency of the original trans-acting TW17 ribozyme system. Our studies indicated that the original trans-acting TW17 ribozyme system had satisfactory binding affinity with Kd of 0.18 ?b 0.06 ?嵱. When strengthening the binding stem between the two RNA moieties or extending their binding stem length, these structural-modified trans-acting TW17 ribozyme systems showed similar or inferior binding affinity (similar or higher Kd values) to that of the original one. Adsorption of the substrate RNA on the catalytic core RNA is thus unlikely the rate-determining step during catalysis. This study sheds light on the mechanism of TW17 ribozyme catalysis.
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author2 |
Tzu-Pin Wang |
author_facet |
Tzu-Pin Wang Yi-Ming Liou 劉宜明 |
author |
Yi-Ming Liou 劉宜明 |
spellingShingle |
Yi-Ming Liou 劉宜明 Studies of Trans-Acting TW17 ribozyme Catalysis Systems to Decipher Its Phosphorothiolate Thiolester Hydrolysis Mechanism |
author_sort |
Yi-Ming Liou |
title |
Studies of Trans-Acting TW17 ribozyme Catalysis Systems to Decipher Its Phosphorothiolate Thiolester Hydrolysis Mechanism |
title_short |
Studies of Trans-Acting TW17 ribozyme Catalysis Systems to Decipher Its Phosphorothiolate Thiolester Hydrolysis Mechanism |
title_full |
Studies of Trans-Acting TW17 ribozyme Catalysis Systems to Decipher Its Phosphorothiolate Thiolester Hydrolysis Mechanism |
title_fullStr |
Studies of Trans-Acting TW17 ribozyme Catalysis Systems to Decipher Its Phosphorothiolate Thiolester Hydrolysis Mechanism |
title_full_unstemmed |
Studies of Trans-Acting TW17 ribozyme Catalysis Systems to Decipher Its Phosphorothiolate Thiolester Hydrolysis Mechanism |
title_sort |
studies of trans-acting tw17 ribozyme catalysis systems to decipher its phosphorothiolate thiolester hydrolysis mechanism |
publishDate |
2011 |
url |
http://ndltd.ncl.edu.tw/handle/54991295890093656380 |
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