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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

基本信息

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

项目摘要

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（Cap-binding protein，简称CBP或IF-4E）在活细胞中含量极低，对启动核糖体上的蛋白质合成起重要作用。为了阐明该蛋白的生化和结构性质，明确其对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)成功化学合成了人类帽结合蛋白（hCBP）基因（总碱基对660对）。(2)在pBR322载体中构建了该基因的包涵体，并以hCBP作为包涵体大量表达人生长激素。(3)建立了hCBP与混淆蛋白的分离及HPLC纯化方法。(4)通过一系列模型相互作用研究，研究了可能在7-甲基鸟嘌呤碱基（m7G） mRNA帽结构选择性识别中起作用的功能氨基酸残基。(5)基于上述认识，通过定点诱变将hCBP的功能氨基酸转化为非功能氨基酸。建立了基因突变和突变体hCBP蛋白的分离纯化方法。(6)化学合成了一系列mRNA cap类似物。(7)通过紫外光谱和荧光光谱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。结果表明，His-33、His-37、Trp-102和Glu-105残基在与mRNA帽结构的相互作用中起重要作用。此外，含m7g的柱与原生和突变hCBPs的亲和力结合实验也得到了相同的结果。(8) x射线晶体和核磁共振溶液分析正在进行中。