| Item type |
学位論文 / Thesis or Dissertation(1) |
| 公開日 |
2010-02-22 |
| タイトル |
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タイトル |
UV-sensor motifs in promoters |
| タイトル |
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タイトル |
UV-sensor motifs in promoters |
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言語 |
en |
| 言語 |
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言語 |
eng |
| 資源タイプ |
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資源タイプ識別子 |
http://purl.org/coar/resource_type/c_46ec |
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資源タイプ |
thesis |
| 著者名 |
宮本, 貴史
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| フリガナ |
ミヤモト, タカシ
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| 著者 |
MIYAMOTO, Takashi
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| 学位授与機関 |
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学位授与機関名 |
総合研究大学院大学 |
| 学位名 |
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学位名 |
博士（理学） |
| 学位記番号 |
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内容記述タイプ |
Other |
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内容記述 |
総研大甲第1166号 |
| 研究科 |
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|
値 |
生命科学研究科 |
| 専攻 |
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値 |
18 遺伝学専攻 |
| 学位授与年月日 |
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学位授与年月日 |
2008-03-19 |
| 学位授与年度 |
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値 |
2007 |
| 要旨 |
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内容記述タイプ |
Other |
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内容記述 |
It is well known that transcription elongation and repair of UV－damage is coupled.<br />In eukaryotes，stalled RNA pol II recruits several repair proteins to from a complex<br />containing RNA pol II，TFIIH，and the repair proteins．In prokaryotes，stalled RNAP<br />recruits Mfd protein．Mfd has a domain homologous to the domain in UvrB which<br />recognizes a wide range of structurally diverse lesions in addition to a domain for<br />interaction with RNAP．Mfd binds to the DNA segment immediately upstream of the<br />stalled RNAP and pushes it towards downstream by using the energy of<br />ATP-hydrolysis to dissociate it from DNA，and speculated to recruit UvrA to perform<br />the repair similar to the global nuclear excision repair. These mechanisms provide the<br />concept on the triggering DNA repair rather than that on transcriptional regulation．<br /> Although RNAP tends to stall at all kinds of UV-damages，the efficiency of stallat at <br />a UV-damage has not been measured．Therefore，it is possible that inhibition of<br />transcription is carried out by a mechanism other than the stall of RNAP．The stall<br />could be overcome by a read-through of the damaged base by misincorporation，<br />slippage，Jump，and switching the template strand．These considerations indicate the<br />mechanism of coulpling of UV-damages and initiation，because initiation generally has<br />the largest dynamic range of transcriptional regulation．Irrespective of the efforts to<br />find specific factors triggering the inhibition in initiation，such factors have not been<br />found yet, suggesting an alternative possibility that the triggering function has been<br />already installed in RNAP．<br /> Transcription initiation is biochemically separated into several steps．RNAP in the<br />form of holoenzyme binds to a promoter and then DNA duplex in the complex is<br />partially unwound to expose the template strand of DNA by mostly unknown<br />mechanism. The resultant complex is called open complex and this complex was once<br />considered to be homogeneous．However，it is now known to involve two or more<br />conformations which have different catalytic properties，forming branched pathway<br />mechanism. One branch leads to produce the full-length transcripts（productive<br />pathway），While another leads to produce only short transcripts iteratively<br />synthesized by“moribund complex”which is defined as a complex that produces only<br />abortive but no full-length transcripts（non-productive pathway）.On some promoters，<br />moribund complex is converted into dead-end complex which still maintains transcript<br />but lacks elongation activity．Accumulation of dead-end complex cause less<br />stoichiometric synthesis of the full-length products in a single-round transcription<br />assay.<br /> To examine the potential coupling and its mechanism，I have studied on the<br />relationship between transcription initiation and UV-damage by using purified<br />reconstitution system of E.coli．This well-characterized system allows me to consider<br />the relationship between the structure and the function．The system is composed of<br />RNAP holoenzyme，a promoter DNA，and four NTPs as substrates．I selected the T7A1<br />promoter because little moribund and dead-end complexes are accumulating on this<br />promoter，simplifying the analysis of the branched pathway. By irradiating the<br />promoter DNA with UV light，UV damages are generated at adjacent pyrimidine bases<br />in the promoter region. If the generated damages are up stream from the transcription<br />start site，such damages are nothing to do with elongation pause and thus their effect<br />on transcription must be limited to the effect on initiation．<br /> In this study, UV-irradiation of the template DNA was found to enhance abortive<br />initiation and to induce dead-end complex，namely enhancing the nonproductive<br />pathway．The positions of UV-damage in the template DNA in moribund and dead-end<br />complex was compared with those in the binary complex that contains productive<br />complex．The pyrimidine dimers at several sites in the upstream from the<br />transcription start site，but not all，were enriched in the moribund and dead-end<br />complexes．indicating that these UV-damaged pyrimidine bases induce these<br />complexes. These positions of the damages are on both template and non-template<br />strands. The induction by the damaged pyrimidine residues are confirmed by the<br />transcription on DNA fragments which contain single UV-damageds in the upstream<br />region．Furthermore，I found that adjacent pyrimidine bases are conserved atone or<br />more positions of those identified on T7A1 promoter among the majority of the E．coli<br />promoters，suggesting that these positions would be UV-sensor motif in promoters．<br /> |
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値 |
有 |
要旨・審査要旨 (270.9 kB)
