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

将兔网织红细胞裂解物与无帽的烟草花叶病毒RNA(TMV-RNA)5‘端70个核苷酸长片段及其3’端的启动子Aug密码子孵育形成80s起始复合体，命名为Ad^&lt；32&gt；P&lt；Omeg&gt；标记的^&lt；32&gt；p&lt；omega&gt；亚片段被80年代启动的核糖体保护，不被微球菌核酸酶消化，并与5SRNA-L5蛋白颗粒(5SRNP)结合。为了确定启动子Aug结合蛋白，用紫外光照射起始复合体(40S和80S)，诱导核糖体蛋白与^&lt；32&gt；p&lt；omega&gt；分析表明，40S起始复合体中含有S7、S10、S25、S29和L5蛋白，80S起始复合体中含有S7、S25、S29和L5蛋白。对40S复合体中低分子量RNA的分析表明，5SRNA稳定地与40S亚基结合。同样的结果，在大鼠肝脏的天然40S亚基中也显示了5SRNA与40S亚基的结合。

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.