CONTROL OF INITIATION FREQUENCY OF COLE2 REPLICATION

COLE2复制起始频率的控制

• 批准号: 08458217
• 负责人: ITOH Tateo
• 金额: $ 4.93万
• 依托单位: SHINSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458217/
• 关键词: ColE2 plasmid; Replication initiator; primer RNA; origin; DNA binding protein; antisense RNA; translation; RNA secondary structure; ColE2プラスミド; 複製開始蛋白質; 過剰複製阻止; コピー数調節; ColE2群プラスミド; 二量体形成; 直列繰り返し配列; RNaseIII; 複製開始頻度

The ColE2 Rep protein is unique among many essential initiator proteins in that it is a plasmid-specified primase specific for initiation of DNA replication in the origin by DNA polymerase I.Expression of the Rep protein is kept constant through negative regulation by a small antisense RNA (RNAI). RNAl is entirely complementary to the 5' nontranslated region of the Rep mRNA.Since the Rep protein is trans-acting, it is not so obvious how the initiation frequency can be kept constant.Analyses of various derivatives of the ColE2 origin carrying deletions or single-base substitutions showed that the region may be divided into three subregions : one important for stable binding of the Rep protein, another important for binding and for replication and another important for replication but apparently not for binding. Transformation frequency of the autonomously replicating plasmids carrying deletions in the first region is lower and nevertheless the copy numbers of them in hostbacteria are higher as compared with the wild-type plasmid. The Rep protein might inhibit over-replication or re-replication of newly replicated daughter molecules by stable binding to the origin. This might be important to keep the initiation frequency at a constant level.The 5' nontranslated region of the Rep mRNA far upstream of the initiation codon and another region in the coding region near the initiation codon are important for efficient expression of the Rep protein. Partial digestion with various RNases in the presence or absence of RNAI suggested that binding of RNAI to the Rep mRNA causes changes in the secondary structure of the region containing the initiation codon. Mutant plasmids carrying base substitutions in the upstream and downstream regions of the initiation codon of the Rep mRNA have been isolated. Some of the mutations abolished or decreased expression of the Rep protein.

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项目成果

Miki Shinohara: "Specificity determinants in interaction of the initiator(Rep)proteins with the origins in the plasmids ColE2-P9 and ColE3-CA38 identified by chimera analysis." J.M.B.257. 290-300 (1996)

Miki Shinohara：“通过嵌合体分析鉴定出启动子 (Rep) 蛋白与质粒 ColE2-P9 和 ColE3-CA38 起源相互作用的特异性决定因素。”

YAGURA,MASARU: "Functional organization of plasmid ColE2 initiator (Rep) protein and replication origin." GENES GENET.SYST.72. 382 (1997)

YAGURA、MASARU：“质粒 ColE2 启动子 (Rep) 蛋白的功能组织和复制起点。”

Miki Shinohara: "Specificity determinants in interaction of the initiator (Rep) proteins with the origins in the plasmids Co1E2-P9 and Co1E3-CA38 identified by chimera analysis." J.M.B.257. 290-300 (1996)

Miki Shinohara：“通过嵌合体分析鉴定出启动子 (Rep) 蛋白与质粒 Co1E2-P9 和 Co1E3-CA38 起源相互作用的特异性决定因素。”

Miki Shinohara: "Specificity determinants in interaction of the initiator (Rep) proteins with the origins in the plasmids ColE2-P9 and ColE3-CA38 identified by chimera analysis." J.M.B.257. 290-300 (1996)

Miki Shinohara：“通过嵌合体分析鉴定出启动子 (Rep) 蛋白与质粒 ColE2-P9 和 ColE3-CA38 起源相互作用的特异性决定因素。”

Masaru Yagura: "Functional prganization of plasmid Co1E2 initiator (Rep) protein and replication origin" Genes Genet.Syst.72. 382 (1997)

Masaru Yagura：“质粒 Co1E2 启动子 (Rep) 蛋白和复制起点的功能组织”Genes Genet.Syst.72。