Sructural and function analysis of yeast mRNA capping enzyme in transcription reaction.

转录反应中酵母mRNA加帽酶的结构和功能分析。

• 批准号: 11680613
• 负责人: SHIBAGAKI Yoshio
• 金额: $ 1.66万
• 依托单位: Kitasato University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680613/
• 关键词: Capping enzyme; Cap structure; S.cerevisiae; in vitro transcription; Transcription factor; in vitro 転写反応

The yeast mRNA capping enzyme is composed of Ceg1 and Cet1, responsible for the activities of mRNA guanylyltransferase (GTase) and RNA 5'-triphosphatase (TPase), respectively. To investigate structure and function of Cet1 , we had isolated and characterized temperature sensituve (ts) mutants. For isolation of cet1^<ts> mutants on single-copy plasmid, YCpW-CET1 was treated with hydroxylamine and transformed into SK1 which is a chromosomal cet1Δ : : LEU2 disruptant carrying with episomal malti-copy plasmid, YEp-CET1 containing wild type CET1. Transformants were picked and grown on FOA (5-fluoroorotic acid) plates at 25℃ to carry out plasmid shuffling and to express mutated Cet1. We isolated 7 temperature-sensitive mutants of CET1 (cet1^<ts>-1 to cet1^<ts>-7). All these mutations located in the essential for TPase activity (265-549) and Cet1-Ceg1 interaction (205-265) regions based on deletion mutation analysis. We expressed all cet1^<ts> mutant proteins as GST fusion in E.coli and assayed these RNA 5'-triphosphatase activity. Three ts mutants, G257D (cet1^<ts>-1), S419L (cet1^<ts>-2), T396I/T400I (cet1^<ts>-3), had enough TPase activity. Contrary to these mutants, R532K mutation made greately reduced TPase activity but not ts mutant. These result indicated that it is different between reduction of TPase and temperature sensitivity. One interesting mutation (R242K) made ts mutant. but R242K/A257N or R242K/E200K double mutation looked growth normal. This data suggested that three amino acids (A257, R242 and E200) were working with Cet1-Ceg1 interaction cooperatively.

酵母 mRNA 加帽酶由 Ceg1 和 Cet1 组成，分别负责 mRNA 鸟苷酸转移酶 (GTase) 和 RNA 5'-三磷酸酶 (TPase) 的活性。为了研究 Cet1 的结构和功能，我们分离并表征了温度敏感 (ts) 突变体。为了在单拷贝质粒上分离cet1^<ts>突变体，YCpW-CET1用羟胺处理并转化到SK1中，SK1是携带附加型麦芽拷贝质粒的染色体cet1Δ::LEU2破坏体，YEp-CET1含有野生型CET1。挑取转化子并在FOA（5-氟乳清酸）平板上于25℃生长以进行质粒改组并表达突变的Cet1。我们分离了 7 个 CET1 温度敏感突变体（cet1^<ts>-1 至 cet1^<ts>-7）。根据缺失突变分析，所有这些突变都位于 TPase 活性必需的 (265-549) 和 Cet1-Ceg1 相互作用 (205-265) 区域。我们在大肠杆菌中将所有 cet1^<ts> 突变蛋白表达为 GST 融合体，并测定了这些 RNA 5'-三磷酸酶活性。三个 ts 突变体 G257D (cet1^<ts>-1)、S419L (cet1^<ts>-2)、T396I/T400I (cet1^<ts>-3) 具有足够的 TPase 活性。与这些突变体相反，R532K突变使TPase活性大大降低，但ts突变体则不然。这些结果表明TPase的减少和温度敏感性之间是不同的。一种有趣的突变（R242K）产生了 ts 突变体。但R242K/A257N或R242K/E200K双突变看起来生长正常。该数据表明三个氨基酸（A257、R242 和 E200）与 Cet1-Ceg1 相互作用协同作用。

项目成果

Fukamachi N., Tsukamoto T., Shibagaki Y., Mizumoto K.: "Interaction between human mRNA capping enzyme and transcription initiation complex of RNA polymerase II."Seikagaku. 71. 953 (1999)

Fukamachi N.、Tsukamoto T.、Shibagaki Y.、Mizumoto K.：“人 mRNA 加帽酶与 RNA 聚合酶 II 转录起始复合物之间的相互作用。”Seikagaku。

柴垣 芳夫: "酵母を用いたmRNAキャッピング酵素の機能解析"生化学. 71・8. 953 (1999)

Yoshio Shibagaki：“使用酵母的mRNA加帽酶的功能分析”生物化学71・8（1999）。

Tsukamoto,T.: "Cloning and Charactrization of three human cDNA encoding mRNA(guanine-7-)methyltransferase, and mRNA cap methlate"Biochem.Biophys.Res.Comm. 251. 27-34 (1998)

Tsukamoto,T.：“编码 mRNA(鸟嘌呤-7-)甲基转移酶和 mRNA 帽甲基化酶的三种人类 cDNA 的克隆和表征”Biochem.Biophys.Res.Comm。

柴垣芳夫: "酵母mRNAキャッピング酵素βサブユニットの機能解析"生化学. 72・8. 979 (2000)

Yoshio Shibagaki：“酵母mRNA加帽酶β亚基的功能分析”生物化学72・8（2000）。

Shibagaki Y., Shigemori S., Kumakubo S., Tsukamoto T., Mizumoto K.: "Functional analysis of yeast mRNA capping enzyme β-subunit"Seikagaku. 72. 979 (2000)

Shibagaki Y.、Shigemori S.、Kumakubo S.、Tsukamoto T.、Mizumoto K.：“酵母 mRNA 加帽酶 β 亚基的功能分析” Seikagaku. 72. 979 (2000)。