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.

通过将兔网织红细胞裂解物与不带帽且其起始子 AUG 密码子位于 3' 末端的烟草花叶病毒 RNA (TMV-RNA) 5' 末端 70 核苷酸长片段（指定为 ad ^）一起孵育，形成 80S 起始复合物。 32P<OMEG>，其用^32pCp标记。经80S起始核糖体保护免受微球菌核酸酶消化的^ 32 P<OMEGA>的标记亚片段附着至5SRNA-L5蛋白颗粒(5SRNP)。为了确定起始子AUG结合蛋白，用UV光照射起始复合物(40S和80S)以诱导核糖体蛋白和β 32 p OMEGA之间的交联。对交联的β 32 p-蛋白的分析表明，40S起始复合物中蛋白质S7、S10、S25、S29和L5，以及80S起始复合物中蛋白质S7、S25、S29和L5。对 40S 复合物中低分子量 RNA 的分析表明，5SRNA 与 40S 亚基稳定相关。在来自大鼠肝脏的天然 40S 亚基中也显示出相同的结果，即 5SRNA 与 40S 亚基的关联。