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

Research Project

Project/Area Number	61570120
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	General medical chemistry
Research Institution	Niigata University
Principal Investigator	TAKAHASHI Yoshiaki Niigata University, Assistant, 医学部, 助手 (60115045)
Co-Investigator(Kenkyū-buntansha)	KENMOCHI Naoya Niigata University, School affairs the faculty, 医学部, 教務職員 (00133124) MISHIMA Yukio Niigata University, Lecturer, 医学部, 講師 (30166003)
Project Period (FY)	1986 – 1987
Project Status	Completed (Fiscal Year 1987)
Budget Amount *help	¥1,900,000 (Direct Cost: ¥1,900,000) Fiscal Year 1987: ¥500,000 (Direct Cost: ¥500,000) Fiscal Year 1986: ¥1,400,000 (Direct Cost: ¥1,400,000)
Keywords	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
Research Abstract	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.