Using fluorescence resonance energy transfer technique to map eukaryotic RNA polymerase elongation complex
碩士 === 臺灣大學 === 微生物與生化學研究所 === 95 === Together with six general transcription factors and promoter DNA, yeast RNA polymerase II (RNA pol II) forms the core enzyme responsible for the synthesis of messenger RNA from genome DNA, a crucial role in eukaryote. The atomic structures of RNA polymerase II e...
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| Format: | Others |
| Language: | zh-TW |
| Published: |
2007
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| Online Access: | http://ndltd.ncl.edu.tw/handle/07426687606754117567 |
| Summary: | 碩士 === 臺灣大學 === 微生物與生化學研究所 === 95 === Together with six general transcription factors and promoter DNA, yeast RNA polymerase II (RNA pol II) forms the core enzyme responsible for the synthesis of messenger RNA from genome DNA, a crucial role in eukaryote. The atomic structures of RNA polymerase II elongation complex have been resolved by using X-ray crystallography technology, but the nascent RNA exit route on the complex remains unknown.
Fluorescence resonance energy transfer (FRET) is a physical tool that can measure intramolecular or intermolecular distance to nanometer precision. In this work, we use a novel strategy that enables us to measure the distance between 5’ end of designed RNA primers and any subunits in the RNA pol II elongation complex. We have successfully determined the position of 5’ end of the synthesized 10mer and 17mer RNA primers and thus mapped the exiting route of RNA from RNA pol II. Furthermore, we successfully used the in gel fluorescence resonance energy transfer (gelFRET) assay to demonstrate the formation of RNA pol II elongation complex. We further use smFRET to measure the distance between 5’ RNA and c-terminal of Rpb3.
Our finding that 10mer RNA assumes higher FRET with the c-terminal of Rpb3 than 17mer RNA suggests the hypothesis RNA exits through a channel toward Rpb7 instead of towards Rpb8.
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