{"created":"2023-06-20T13:20:53.834698+00:00","id":963,"links":{},"metadata":{"_buckets":{"deposit":"9a6c0793-77e6-454e-9133-c9ff3f2b22d5"},"_deposit":{"created_by":1,"id":"963","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"963"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00000963","sets":["2:430:20"]},"author_link":["10032","10033","10034"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"野村, 扶"}],"nameIdentifiers":[{"nameIdentifier":"10032","nameIdentifierScheme":"WEKO"}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"ノムラ, タスク"}],"nameIdentifiers":[{"nameIdentifier":"10033","nameIdentifierScheme":"WEKO"}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2001-03-23"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"The RNA polymerase core enzyme of Escherichia coli with the catalytic activity of RNA synthesis is assembled sequentially under the order: 2α→α<SUB>2</SUB>→α<SUB>2</SUB>β→α2ββ'(premature core)→E (active core; E represents the active form of α<SUB>2</SUB>ββ'). The core enzyme gains the activities of promoter recognition and transcription initiation after binding one of seven molecular species of the σ subunit. The β subunit plays a major role in the catalytic activity of RNA polymerization and provides the binding sites for substrates and nascent RNA. The expression of these intrinsic activities, however, completely depends on the interaction with the α and β' subunits. In order to get further insight into the structure-function relationship of β, I tried in this study to map the protein-protein contact surfaces on β with other core subunits. For the identification of α subunit contact sites, the tryptic cleavage pattern was compared between isolated free β and β in the α<SUB>2</SUB>β complex. Results indicated that the central part of β between the conserved regions E and F were protected from tryptic digestion after binding of the α subunit. In vitro binding assays of various β fragments with the α subunit indicated that two regions, β(737-936) including the regions E and F and β(937-1138) are involved in α binding. These two regions were also shown to be required for binding of β' to the α<SUB>2</SUB>β complex to form the core enzyme.<br />The β subunit of assembled RNA polymerase is involved in moelcular interaction with a group of transcription factors (β-contact or class-III factors), leading to modulation of the specificity and activity of RNA polymerase. In order to identify novel species of the class-III transcription factor, I tried to isolate β-associated proteins from extracts of cells expressing GST (glutathione S-transferase)-tagged β at low levels. Protein complexes containing GST-β were isolated by glutathione affinity column chromatography, and separated by SDS-PAGE. After micro-sequencing and mass-spectroscopy, the candidate proteins of class-III transcription factor have been identified, including HepA, glycerol kinase (GlpK), methionine adenosyl transferase (MATase), YfhO, elongation factor EF-Tu, ribosomal proteins L1, L13 and S6. Binary complex formation test between the purified class-III factor candidates and the RNA polymerase indicated that HepA forms a stable complex with the RNA polymerase core enzyme, but not with the holoenzyme. Six proteins, GlpK, MATase, YfhO, L1, L13 and S6, so far tested did not form stable complexes under the conditions employed. The quantitative Western blot analysis indicated that the HepA protein was synthesized only in the exponential growth phase at the concentration of about one-third the level of σ<SUP>70</SUP> subunit, and declined to an undetectable in the stationary phase.<br />For identification of possible influence of the interplay between HepA and RNA polymerase, the hepA gene was disrupted. The hepA null mutant, however, did not show any growth defect in a rich medium. DNA chip analysis was carried out to detect influence of the hepA disruption on overall gene transcription. A small number of genes were found to be up- or down-regulated in the hepA null mutant, suggesting HepA is involved in transcriptional regulation of a set of E.coli genes.","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534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":"2000"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"NOMURA, Tasuku","creatorNameLang":"en"}],"nameIdentifiers":[{"nameIdentifier":"10034","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","filename":"甲534_要旨.pdf","filesize":[{"value":"196.9 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨 / Abstract, Screening Result","url":"https://ir.soken.ac.jp/record/963/files/甲534_要旨.pdf"},"version_id":"b94a054c-b960-493c-ac7b-6d89f8c147a8"}]},"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":"Protein-protein interactions on the β Subunit of Esherichia coli RNA polymerase","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Protein-protein interactions on the β Subunit of Esherichia coli RNA polymerase"},{"subitem_title":"Protein-protein interactions on the β Subunit of Esherichia coli RNA polymerase","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["20"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"963","relation_version_is_last":true,"title":["Protein-protein interactions on the β Subunit of Esherichia coli RNA polymerase"],"weko_creator_id":"1","weko_shared_id":1},"updated":"2023-06-20T14:41:20.408930+00:00"}