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分裂酵母を用いたＲＮＡポリメラーゼIIの構造解析
https://ir.soken.ac.jp/records/889
https://ir.soken.ac.jp/records/889a574bba2-5ee0-4fb1-b5bc-285ec27d3308
| 名前 / ファイル | ライセンス | アクション |
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要旨・審査要旨 / Abstract, Screening Result (354.2 kB)
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本文 (3.0 MB)
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| Item type | 学位論文 / Thesis or Dissertation(1) | |||||
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| 公開日 | 2010-02-22 | |||||
| タイトル | ||||||
| タイトル | 分裂酵母を用いたＲＮＡポリメラーゼIIの構造解析 | |||||
| タイトル | ||||||
| タイトル | Structural Studies of Schizosaccharomyces pombe ＲＮＡ polymerase II | |||||
| 言語 | en | |||||
| 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 |
東, 慶直
× 東, 慶直 |
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| フリガナ |
アズマ, ヨシナオ
× アズマ, ヨシナオ |
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| 著者 |
AZUMA, Yoshinao
× AZUMA, Yoshinao |
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| 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83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 | 1994-03-24 | |||||
| 学位授与年度 | ||||||
| 値 | 1993 | |||||
| 要旨 | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | The molecular composition of DNA‐dependent RNA polymerase II from Schizosaccharomyces pombe was studied.First,RNA polymerase II was purified from the wild type S.pombe, in five steps:precipitation with Polymin P;elution from the Polymin P precipitates; separation of RNA polymerase II from RNA polymerases I and III by DEAE-Sephadex column chromatography; purification by Q Sepharose FF; and by Superose 6 gel filtration column chromatogfaphy.The highly purified S.pombe RNA polymerase II contained more than eleven polypeptides as analyzed by SDS-po1yacrylamide gel electrophoresis.The molar ratio of the three large subunit,RPB1,2 and 3,was 1:1:2,which is identical with that of the E.coli and S.cerevisiae enzymes.<br /> In order to establish in vitro reconstitution system of RNA polymerase II and to decide the subunit composition of RNA polymerase II,preliminary attempts were made to find conditions for reversible denaturation of RNA polymerase II by treatment with urea.RNA polymerase II was not inactivated up to 0.5M urea.RNA polymerase II 1ost activity from 1.0M to 2.5M urea,but the activity was recovered perfectly simply by dilution of urea. Above 3.0M urea,howevef,RNA polymerase II activity was irreversibly inactivated.From the result of gel filtration,multi‐subunit complexes of RNA polymerase II were observed but several small components were missing from the complexes after denaturation in 4M urea.<br /> The S.pombe gene coding for the largest subunit(subunit 1) of RNA polymerase II was cloned using the S.cerevisiae corresponding gene,RPB1,as a probe in cross‐hybridization.The sequence determination of both the entire genomic DNA and parts of cDNA indicated that this rpb1 gene has six introns in the N-terminal region and encodes the subunit 1 of 1,752 amino acid residues with the molecular mass of 194kDa.From Southern analysis and gene disruption experiments,it was found that this rpb1 gene exists as a single copy in the S.pombe genome and is essential for cell viability. Northern analysis and sequence determination of 3´-and 5´-terminal regions of rpb1 transcript indicated that the size of the rpb1 transcript is about 5.6kb in length.<br /> Among the subunit 1 of S,pombe RNA polymerase II and other b´homo1ogues,nine structurally conserved domains(domain A to H and CTD)were identified:domain A,aputative zinc‐binding site with the consensus sequence of CX2CX9HX2H;domains C and D,the conserved sequences within E.coli DNA polymerase I and T7 DNA polymerase;Domain C,a single two‐helix motif for putative DNA binding:domain F,the putative a‐amanitin binding site:CTD,highly conserved unique repetition with the unit sequence of YSPTSPS among the largest subunits of RNApolymerase II.ln the subunit 1 of S.pombe RNA polymerase II,29 repeats exist in CTD.<br /> The cDNA fragment coding for the third largest subunit(subunit 3) of S.pombe RNA polymerase II was cloned by RT‐PCR using primers designed from the amino acid sequences of V8 fragments of subunit 3.A genomic DNA fragment carrying the entire subunit 3 gene(rpb3) was isolated by hybridization using this cDNA fragment as a probe.The sequence determination indicated that the coding frame of rpb3 is interrupted by two introns and this gene encodes subunit 3 of 297 amino acids in length.Southern and Nothern analyses indicated that the rpb3 gene is present as a single copy in haploid S.pombe cells and the size of rpb3 transcript is about 1.2kb in length.<br /> Among the RNApolymerase subunit 3 from various organisms,four structural conserved domains(domains A to D) were found:domains A and D exist even in the a subunit of E.coli RNA polymerase; and domains B and C are conserved only in eukaryotic RNA polymerases.<br /> Domain A may play a role in subunit‐subunit contact of RNA polymerase.Domain B with a putative meta1‐binding sequence,CXCX3CX2C,exists only in RNA polymerase II,but not in RNA polymerase I nor III.Domain D with a leucine zipper-like motif may be required for the formation and/or stability of RNA polymerase.Subunit 3 of eukaryotic RNA polymerases lacks the sequence corresponding to the C‐terminal region of E.coli RNA polymerase a subunit carrying the contact site I for some transcription activators.<br /> To identify the function(s) of subunit 3,the rpb3 gene was mutagenized by lower fidelity PCR and transferred into S.pombe using either spheroplast method or electroporation method.<br /> Total 178 temperature‐sensitive ts mutants were isolated from about nine thousand transformants. PCR analysis and Southern analysis were carried out for 68 stable ts mutants.Most of the mutants tested carried a single copy of full length DNA fragment integrated in the genomic DNA,With nine mutants,the mutation sites were determined after cloning and sequencing.All the mutant rpb3 genes carried multiple mutations,but many mutations were clusteredin the N-terminal region of RPB3 polypeptides.Upon temperaturc shift from permissive temperaturc,25-C to non‐permissive temperature,37-C,some of the mutants stop growing immediately,while other mutants stop growing s1owly,implying that the assembly of RNA polymerase II is defective in these mutants.Some mutants show abnormal cell shapes(fof example,7 times 1onger in cell size) at the non‐permissive temperature.None of the sixteen mutants survived after one day incubation at non‐permissive temperature.Even in these non‐viable cells,the nuclei still appeared intact. | |||||
| 所蔵 | ||||||
| 値 | 有 | |||||
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| 内容記述タイプ | Other | |||||
| 内容記述 | application/pdf | |||||
