Expression of mRNA cap-binding protein and structure analysis of its cap recognition mechanism

帽结合蛋白mRNA的表达及其帽识别机制的结构分析

• 批准号: 02453146
• 负责人: ISHIDA Toshimasa
• 金额: $ 3.07万
• 依托单位: Osaka University of Pharmaceutical Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02453146/
• 关键词: cap-binding protein; IF-4E; mRNA cap structure; gene expression; recognition; structure analysis; IFー4E

Cap-binding protein (named CBP or IF-4E), the content of which is very small amount in living cell, plays an important role in initiating the protein synthesys on ribosome. In order to elucidate the biochemical and structural nature of this protein and to make clear the selective recognition of mRNA cap structure, we carried out this research project. The main results obtained so far are as follows:(1) We succeeded in the chemical synthsis of gene of human cap-binding protein (hCBP)(total base pairs of 660).(2) We established the inclusion of this gene into pBR322 vector and the large amount of expression of hCBP as the inclusion body with human growth hormone.(3) We established the separation of hCBP from the confused protein and the HPLC purification method.(4) The functional amino acid residues which probably functions in the selective recognition for the mRNA cap structure characterized by 7-methylguanine base (m7G) were surveyed by a series of model interaction studies.(5) Based on the above insight, the functional amino acids of hCBP were transformed to the non-functional amino acids by the site-directed mutagenesis. The methods for the gene mutations and the isolation and purification of mutant hCBP proteins were established.(6) A series of mRNA cap analogues were chemically synthesized.(7) The interaction modes of these cap analogues with the native and mutant hCBP proteins were investigated by UV and fluorescence spectroscopies. As a result, it was made clear that the His-33, His-37, Trp-102 and Glu-105 residues play important role in interaction with mRNA cap structure. Further, the same results were obtained from the binding experiments of the affinity of m7G-containing column with native and mutant hCBPs.(8) The X-ray crystal and NMR solution analyses are now in progress.

帽结合蛋白(CBP或IF-4E)在活细胞中含量极少，在启动核糖体蛋白质合成中起重要作用。为了阐明该蛋白的生化和结构性质，明确mRNA帽结构的选择性识别，我们开展了本研究项目。目前取得的主要成果如下：(1)成功地化学合成了人帽结合蛋白(hCBP)基因(总碱基对660个)。(2)建立了该基因在pBR322中的包涵体。 (3)建立了hCBP从混淆蛋白中的分离及HPLC纯化方法。(4)推测了hCBP可能在其中发挥作用的功能性氨基酸残基。通过一系列模型相互作用研究，对以7-甲基鸟嘌呤碱基（m7G）为特征的mRNA帽结构进行选择性识别。（5）基于上述见解，将hCBP的功能性氨基酸转化为非功能性氨基酸通过定点诱变。建立了突变hCBP蛋白的基因突变和分离纯化方法。(6)化学合成了一系列mRNA帽类似物。(7)研究了这些帽类似物与天然和突变hCBP蛋白的相互作用模式。通过紫外和荧光光谱。结果表明，His-33、His-37、Trp-102和Glu-105残基在与mRNA帽子结构的相互作用中发挥重要作用。此外，含m7G的柱与天然和突变hCBP的亲和力结合实验也得到了相同的结果。(8)X射线晶体和NMR溶液分析正在进行中。

项目成果

T.Ishida: "Molecular design of oligopeptides possessing the binding ability selective for a target nucleic acid base" Advances in Pharmaceutical Sciences. 8. 55-81 (1992)

T.Ishida：“具有选择性靶核酸碱基结合能力的寡肽的分子设计”药物科学进展。

H.Ueda: "Eur.J.Biochem." Cooperative Stacking and Hydrogen Bond Pairing Interaction of Fragment Peptide in Cap Binding Protein with mRNA Cap Structure,

H.Ueda：“Eur.J.Biochem”。

Y.Kafuku, Y.Matsui, J.Ohtani, Y.Usami, H.Ueda, M.Doi, M.Inoue, T.Ishida: "Spectroscopic study on interaction of nucleic acid base with tryptophan-containing tripeptides: Acetyl-Trp-X-Trp-" Chem.Pharm.Bull. 39. 2487-2490 (1991)

Y.Kafuku、Y.Matsui、J.Ohtani、Y.Usami、H.Ueda、M.Doi、M.Inoue、T.Ishida：“核酸碱基与含色氨酸三肽相互作用的光谱研究：乙酰色氨酸

H.Ueda: "Combination of Trp and Glu residues for recognition of mRNA cap structure:Analysis of m7G base recognition site of human cap binding protein (IF-4E) by siteOdirected mutagenesis" FEBS Lett.280. 207-210 (1991)

H.Ueda：“Trp 和 Glu 残基的组合用于识别 mRNA 帽结构：通过位点定向诱变分析人帽结合蛋白 (IF-4E) 的 m7G 碱基识别位点”FEBS Lett.280。

T.Ishida, H.Iyo, H.Ueda, M.Doi, M.Inoue: "Selective binding of guanine base by a tryptophan-containing dipeptide" J.Chem.Soc.Chem.Commun. 217-218 (1990)

T.Ishida、H.Iyo、H.Ueda、M.Doi、M.Inoue：“含色氨酸的二肽选择性结合鸟嘌呤碱基”J.Chem.Soc.Chem.Commun。