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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃人則(Joseph Jen Tse Huang) | |
| dc.contributor.author | Jiří Koubek | en |
| dc.contributor.author | 谷喬治 | zh_TW |
| dc.date.accessioned | 2021-06-16T03:57:32Z | - |
| dc.date.available | 2016-02-04 | |
| dc.date.copyright | 2015-02-04 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-12-03 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55342 | - |
| dc.description.abstract | Ensuring labeling specificity for fluorophore labeling is a challenge in fluorescent spectroscopy. This biophysical technique is one of the primary research tools for studying cotranslational protein folding which studies protein conformation before it has been released from the ribosome. Due to the nature of ribosome-bound nascent chains (RNCs), fluorescent labeling must be coupled with translation during which tRNA acts as the carrier of fluorescent amino acid. In this work, a novel overexpressed suppressor tRNAcysAmber is developed for the production of BODIPY FL-labeled RNCs. In vitro transcription as well as overexpression is tested as the methods of suppressor tRNA production. In order to simplify the purification procedures, Bacillus subtilis tRNAcysAmber has been selected for its distinctive sequence from any endogenous E. coli RNA. In a single purification step, ample amounts of tRNAcysAmber have been obtained. As cysteinyl-tRNA synthetase was previously shown to aminoacylate tRNAcysAmber with low efficiency, several point mutations were introduced into the C-terminus of cysteinyl-tRNA synthetase to compensate for the Amber mutation in the tRNA anticodon loop. Out of the cysteinyl-tRNA synthetase mutants, D436S mutant is shown with improved aminoacylation efficiency and specificity towards tRNAcysAmber. In addition, overexpressed BODIPY FL-cysteinyl-tRNAcysAmber indicates improved stability of this tRNA compared to the in vitro transcribed tRNA. Applying this tRNA, the dynamics of single-labeled RNCs by time-resolved anisotropy was studied to reveal information about the protein folding on the ribosome. The natively unfolded phosphorylated insulin receptor domain (PIR) and the zinc-induced folding in zinc-finger RNC help to correlate the fluorescence correlation time with different nascent chain movements. To further study the impact of chaperones on the RNC dynamics at different stages of translation, Entner-Douderoff aldolase (Eda) RNCs with four predetermined chain length are generated in either the wild-type or chaperone-depleted cell-free system. By applying BODIPY FL-cysteinyl-tRNAcysAmber, our results indicate that Eda may start folding without chaperones after approximately half of the protein emerges from the ribosome. In addition, chaperones increase the nascent chain confinement in the full length Eda RNC, which may cause the decrease the binding of the trigger factor, a co-translational chaperone, due to growing hindrances between RNCs and chaperones. Overall, the facile preparation of suppressor tRNA for labeling with fluorophores is demonstrated together with the application of single-residue labeled nascent chains in studying the effect of chaperones on the RNC dynamics. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T03:57:32Z (GMT). No. of bitstreams: 1 ntu-103-D99223212-1.pdf: 9987820 bytes, checksum: 5968e31077014264a7e7777a1c1ea4e7 (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | Abstract i
Acknowledgement iii List of Figures vi List of Tables x Abbreviations xi Introduction 1 Chapter 1: Design and preparation of cysteinyl-tRNA synthetase 6 Materials and methods 9 General 9 Preparation of the cysteinyl-tRNA synthetase 10 Active site titration assay 10 Chapter 2: Preparation of tRNA 12 In vitro transcription 12 Preparation of transcription templates 16 Monitoring RNA transcription optimization 19 Purification of transcribed tRNA 22 tRNA overexpression 31 Overview of in vitro transcription and overexpression for suppressor tRNA production 36 Materials and methods 37 Preparation of transcription templates 37 Screening of transcription conditions 39 RNA transcription, purification and analysis of products 39 Overexpression of Bsu tRNA and isolation of small RNA pool 40 Isolation of Bsu tRNA 41 Chapter 3: Aminoacylation and labeling of tRNA and proteins 42 Testing of cysteinyl-tRNA synthetase mutants 42 Labeling of cysteinyl-tRNAcysAmber 43 Stability of BODIPY FL-cysteinyl-tRNAcysAmber in cell extract 45 Stop-codon suppression by BODIPY FL-cysteinyl-tRNAcysAmber 46 Materials and methods 51 Aminoacylation assay 51 Preparation of BODIPY FL-cysteinyl-tRNA 52 Testing of BODIPY FL-cysteinyl-tRNAcysAmber stability 52 Chapter 4: Time-resolved fluorescence anisotropy of BODIPY FL labeled RNCs 53 Anisotropy analysis of two model RNCs 56 Monitoring the Eda RNCs dynamics in absence of chaperones 62 The influence of chaperones on Eda RNCs dynamics 70 Materials and methods 75 Preparation of template DNA for cell-free reaction 75 Preparation of cell-free system 75 Production of RNCs 76 Time-resolved fluorescence anisotropy measurement 77 Conclusions 78 Appendix I: Theory of fluorescence anisotropy 80 Appendix II: Used nucleotides 86 References 88 | |
| dc.language.iso | en | |
| dc.subject | RNA purification | zh_TW |
| dc.subject | in vitro translation | zh_TW |
| dc.subject | chaperones | zh_TW |
| dc.subject | Protein labeling | zh_TW |
| dc.subject | co-translational protein folding | zh_TW |
| dc.subject | time-resolved fluorescence anisotropy | zh_TW |
| dc.subject | tRNA | zh_TW |
| dc.title | Development of cysteine-specific tRNAs for site-specific protein fluorescence labeling and co-translational protein folding studies | zh_TW |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.coadvisor | 陳平(Richard Ping Cheng) | |
| dc.contributor.oralexamcommittee | 陳長謙(Sunney I. Chan),陳佩燁(Rita P.-Y. Chen),徐尚德(Shang-Te Danny Hsu),劉扶東(Fu-Tong Liu) | |
| dc.subject.keyword | Protein labeling,tRNA,time-resolved fluorescence anisotropy,co-translational protein folding,chaperones,in vitro translation,RNA purification, | zh_TW |
| dc.relation.page | 97 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-12-03 | |
| dc.contributor.author-college | 理學院 | zh_TW |
| dc.contributor.author-dept | 化學研究所 | zh_TW |
| 顯示於系所單位： | 化學系 | |
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