Identification of initiator AUG binding proteins in initiation step of protein synthesis

蛋白质合成起始步骤中起始子 AUG 结合蛋白的鉴定

• 批准号: 61570120
• 负责人: TAKAHASHI Yoshiaki
• 金额: $ 1.22万
• 依托单位: Niigata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-61570120/
• 关键词: Initiation of protein synthesis; Initiator AUG codon; Rabbit reticulocyte lysate; TMV-RNA; 5SRNP; 5SRNP; 80S開始複合体; 開始AUG配列; 開始複合体; リボソーム蛋白; 紫外線照射; Ω断片; 架橋

An 80S initiation complex was formed by incubation of a rabbit reticulocyte lysate with 5'terminal 70-nucleotide-long fragment of tabbaco mosaic virus RNA (TMV-RNA) without the cap and with its initiator AUG codon at 3'terminus, designated ad ^<32>P<OMEG>, which was labeled with ^<32>pCp. Labeled subfragments of ^<32>P<OMEGA>, which were protected from micrococcal nuclease digestion by 80S initiating ribosomes, were attached to 5SRNA-L5 protein particle (5SRNP). To determine initiator AUG binding proteins, initiation complexes (40S and 80S) were irradiated by UV light to induce crosslinks between ribosomal proteins and ^<32>p<OMEGA>. Analyses of the crosslinked ^<32>p-proteins showed that proteis S7, S10, S25, S29 and L5 in 40S initiation complex, and proteins S7, S25, S29 and L5 in 80S initiation complex. Analyses of a low molecular weight RNA in the 40S complex showed that 5SRNA was stably associated to the 40S subunits. The same result, association of 5SRNA to 40S subunits, was shown in native 40S subunits from liver of rat.

将兔网织红细胞裂解物与烟草花叶病毒RNA（TMV-RNA）5 '端70个核苷酸长的片段（不带帽，3'端有起始AUG密码子，命名为ad ^ P，标记为^ pCp）孵育，形成80 S起始复合物<32><OMEG><32>。将标记的β P亚片段<32><OMEGA>连接到5SRNA-L5蛋白颗粒（5SRNP）上，这些亚片段被80 S起始核糖体保护而不被微球菌核酸酶消化。为了确定起始剂AUG结合蛋白，通过UV光照射起始复合物（40 S和80 S）以诱导核糖体蛋白和13 P之间的交联<32><OMEGA>。交联<32>蛋白分析表明，40 S起始复合物中的蛋白质为S7、S10、S25、S29和L5，80 S起始复合物中的蛋白质为S7、S25、S29和L5。对40 S复合物中低分子量RNA的分析表明，5SRNA与40 S亚基稳定结合。同样的结果，5SRNA与40 S亚基在大鼠肝脏中的天然40 S亚基中也显示了缔合。