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These mechanisms provide the\u003cbr /\u003econcept on the triggering DNA repair rather than that on transcriptional regulation.\u003cbr /\u003e Although RNAP tends to stall at all kinds of UV-damages,the efficiency of stallat at \u003cbr /\u003ea UV-damage has not been measured.Therefore,it is possible that inhibition of\u003cbr /\u003etranscription is carried out by a mechanism other than the stall of RNAP.The stall\u003cbr /\u003ecould be overcome by a read-through of the damaged base by misincorporation,\u003cbr /\u003eslippage,Jump,and switching the template strand.These considerations indicate the\u003cbr /\u003emechanism of coulpling of UV-damages and initiation,because initiation generally has\u003cbr /\u003ethe largest dynamic range of transcriptional regulation.Irrespective of the efforts to\u003cbr /\u003efind specific factors triggering the inhibition in initiation,such factors have not been\u003cbr /\u003efound yet, suggesting an alternative possibility that the triggering function has been\u003cbr /\u003ealready installed in RNAP.\u003cbr /\u003e Transcription initiation is biochemically separated into several steps.RNAP in the\u003cbr /\u003eform of holoenzyme binds to a promoter and then DNA duplex in the complex is\u003cbr /\u003epartially unwound to expose the template strand of DNA by mostly unknown\u003cbr /\u003emechanism. The resultant complex is called open complex and this complex was once\u003cbr /\u003econsidered to be homogeneous.However,it is now known to involve two or more\u003cbr /\u003econformations which have different catalytic properties,forming branched pathway\u003cbr /\u003emechanism. One branch leads to produce the full-length transcripts(productive\u003cbr /\u003epathway),While another leads to produce only short transcripts iteratively\u003cbr /\u003esynthesized by“moribund complex”which is defined as a complex that produces only\u003cbr /\u003eabortive but no full-length transcripts(non-productive pathway).On some promoters,\u003cbr /\u003emoribund complex is converted into dead-end complex which still maintains transcript\u003cbr /\u003ebut lacks elongation activity.Accumulation of dead-end complex cause less\u003cbr /\u003estoichiometric synthesis of the full-length products in a single-round transcription\u003cbr /\u003eassay.\u003cbr /\u003e To examine the potential coupling and its mechanism,I have studied on the\u003cbr /\u003erelationship between transcription initiation and UV-damage by using purified\u003cbr /\u003ereconstitution system of E.coli.This well-characterized system allows me to consider\u003cbr /\u003ethe relationship between the structure and the function.The system is composed of\u003cbr /\u003eRNAP holoenzyme,a promoter DNA,and four NTPs as substrates.I selected the T7A1\u003cbr /\u003epromoter because little moribund and dead-end complexes are accumulating on this\u003cbr /\u003epromoter,simplifying the analysis of the branched pathway. By irradiating the\u003cbr /\u003epromoter DNA with UV light,UV damages are generated at adjacent pyrimidine bases\u003cbr /\u003ein the promoter region. If the generated damages are up stream from the transcription\u003cbr /\u003estart site,such damages are nothing to do with elongation pause and thus their effect\u003cbr /\u003eon transcription must be limited to the effect on initiation.\u003cbr /\u003e In this study, UV-irradiation of the template DNA was found to enhance abortive\u003cbr /\u003einitiation and to induce dead-end complex,namely enhancing the nonproductive\u003cbr /\u003epathway.The positions of UV-damage in the template DNA in moribund and dead-end\u003cbr /\u003ecomplex was compared with those in the binary complex that contains productive\u003cbr /\u003ecomplex.The pyrimidine dimers at several sites in the upstream from the\u003cbr /\u003etranscription start site,but not all,were enriched in the moribund and dead-end\u003cbr /\u003ecomplexes.indicating that these UV-damaged pyrimidine bases induce these\u003cbr /\u003ecomplexes. These positions of the damages are on both template and non-template\u003cbr /\u003estrands. The induction by the damaged pyrimidine residues are confirmed by the\u003cbr /\u003etranscription on DNA fragments which contain single UV-damageds in the upstream\u003cbr /\u003eregion.Furthermore,I found that adjacent pyrimidine bases are conserved atone or\u003cbr /\u003emore positions of those identified on T7A1 promoter among the majority of the E.coli\u003cbr /\u003epromoters,suggesting that these positions would be UV-sensor motif in promoters.\u003cbr /\u003e", "subitem_description_type": "Other"}]}, "item_1_description_7": {"attribute_name": "学位記番号", "attribute_value_mlt": [{"subitem_description": "総研大甲第1166号", "subitem_description_type": "Other"}]}, "item_1_select_14": {"attribute_name": "所蔵", "attribute_value_mlt": [{"subitem_select_item": "有"}]}, "item_1_select_8": {"attribute_name": "研究科", "attribute_value_mlt": [{"subitem_select_item": "生命科学研究科"}]}, "item_1_select_9": {"attribute_name": "専攻", "attribute_value_mlt": [{"subitem_select_item": "18 遺伝学専攻"}]}, "item_1_text_10": {"attribute_name": "学位授与年度", "attribute_value_mlt": [{"subitem_text_value": "2007"}]}, "item_creator": {"attribute_name": "著者", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "MIYAMOTO, Takashi", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "0", "nameIdentifierScheme": "WEKO"}]}]}, "item_files": {"attribute_name": "ファイル情報", "attribute_type": "file", "attribute_value_mlt": [{"accessrole": "open_date", "date": [{"dateType": "Available", "dateValue": "2016-02-17"}], "displaytype": "simple", "download_preview_message": "", "file_order": 0, "filename": "甲1166_要旨.pdf", "filesize": [{"value": "270.9 kB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensetype": "license_11", "mimetype": "application/pdf", "size": 270900.0, "url": {"label": "要旨・審査要旨", "url": "https://ir.soken.ac.jp/record/1048/files/甲1166_要旨.pdf"}, "version_id": "dd385290-e2f5-4420-8df4-e7773081ebce"}]}, "item_language": {"attribute_name": "言語", "attribute_value_mlt": [{"subitem_language": "eng"}]}, "item_resource_type": {"attribute_name": "資源タイプ", "attribute_value_mlt": [{"resourcetype": "thesis", "resourceuri": "http://purl.org/coar/resource_type/c_46ec"}]}, "item_title": "UV-sensor motifs in promoters", "item_titles": {"attribute_name": "タイトル", "attribute_value_mlt": [{"subitem_title": "UV-sensor motifs in promoters"}, {"subitem_title": "UV-sensor motifs in promoters", "subitem_title_language": "en"}]}, "item_type_id": "1", "owner": "1", "path": ["20"], "permalink_uri": "https://ir.soken.ac.jp/records/1048", "pubdate": {"attribute_name": "公開日", "attribute_value": "2010-02-22"}, "publish_date": "2010-02-22", "publish_status": "0", "recid": "1048", "relation": {}, "relation_version_is_last": true, "title": ["UV-sensor motifs in promoters"], "weko_shared_id": -1}
UV-sensor motifs in promoters
https://ir.soken.ac.jp/records/1048
https://ir.soken.ac.jp/records/10482adb824e-bfd5-4c2a-becb-da217db3c8b1
名前 / ファイル | ライセンス | アクション |
---|---|---|
![]() |
Item type | 学位論文 / Thesis or Dissertation(1) | |||||
---|---|---|---|---|---|---|
公開日 | 2010-02-22 | |||||
タイトル | ||||||
タイトル | UV-sensor motifs in promoters | |||||
タイトル | ||||||
言語 | en | |||||
タイトル | UV-sensor motifs in promoters | |||||
言語 | ||||||
言語 | eng | |||||
資源タイプ | ||||||
資源タイプ識別子 | http://purl.org/coar/resource_type/c_46ec | |||||
資源タイプ | thesis | |||||
著者名 |
宮本, 貴史
× 宮本, 貴史 |
|||||
フリガナ |
ミヤモト, タカシ
× ミヤモト, タカシ |
|||||
著者 |
MIYAMOTO, Takashi
× MIYAMOTO, Takashi |
|||||
学位授与機関 | ||||||
学位授与機関名 | 総合研究大学院大学 | |||||
学位名 | ||||||
学位名 | 博士(理学) | |||||
学位記番号 | ||||||
内容記述タイプ | Other | |||||
内容記述 | 総研大甲第1166号 | |||||
研究科 | ||||||
値 | 生命科学研究科 | |||||
専攻 | ||||||
値 | 18 遺伝学専攻 | |||||
学位授与年月日 | ||||||
学位授与年月日 | 2008-03-19 | |||||
学位授与年度 | ||||||
2007 | ||||||
要旨 | ||||||
内容記述タイプ | Other | |||||
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 /> | |||||
所蔵 | ||||||
値 | 有 |