Preparation and characterization of eukaryotic initiation factor EIF-3. Formation of binary (EIF-3-Met-tRNAf) and ternary (EIF-3-Met-tRNAf-GTP) complexes.
Preparation and characterization of eukaryotic initiation factor EIF-3. Formation of binary (EIF-3-Met-tRNAf) and ternary (EIF-3-Met-tRNAf-GTP) complexes.
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真核起始因子EIF-3的制备和表征。
DOI：
10.1016/s0021-9258(17)33636-0
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发表时间：
1976
期刊：
影响因子：
--
通讯作者：
I. Wool
中科院分区：
文献类型：
--
作者：
R. S. Ranu;I. Wool
在20 mM KCl中从腹水细胞制备的133，000 Xg上清液级分（低CK 1上清液）含有起始因子EIF-1和EIF-2（以及延伸因子EF-1和EF-2），但缺乏EIF-3;因此，低KCl上清液可用于测定EIF-3。EIF-3是从起始因子粗品（腹水细胞核糖体的250 mM KCl提取物用70%饱和硫酸铵沉淀）经DEAE-Sephadex A-50和羟基磷灰石层析制备的。EIF-O没有可检测到的EIF-1和很少或没有EIF-2。EIF-3是脑心肌炎病毒RNA翻译所必需的因子。通过Sephadex G-200过滤估计EIF-3的分子量为139，000;计算沉降系数约为5.8。EIF-3特异性地与起始tRNA Met-tRNAf形成二元复合物，并且如果存在GTP，则该因子形成三元复合物（EIF-3-Met-tRNAf-GTP）。EIF-3制剂没有甲硫氨酰-tRNA合成酶活性来解释结合。复合物的形成与真核Met-tRNAf和没有其他氨酰-tRNA。二元和三元复合物定量保留在Millipore过滤器（这是最方便的测定），但它们也可以通过Sephadex G-100过滤或甘油梯度离心来证明。GTP增加复合物形成的速率、量和稳定性;在三元复合物中GTP与Met-tRNAf的比例似乎为1。二元复合物和三元复合物将Met-tRNAf转移到40 S核糖体亚基，但不转移到60 S亚基。Met-tRNAf与40 S亚基的因子依赖性结合不需要mRNA（或GTP）。在存在60 S亚基的情况下，即使加入mRNA，与40 S亚基结合的起始tRNA也不会转移到80 S核糖体-该反应可能需要另一个起始因子。用N-乙基马来酰亚胺处理EIF-3导致其在复合物形成和支持脑心肌炎病毒RNA翻译中的活性丧失。除了形成二元和三元复合物，并支持脑心肌炎病毒RNA的翻译外，EIF-3还通过阻止它们的结合或引起80 S对的解离来增加游离核糖体亚基的数量。
The 133,000 X g supernatant fraction prepared from ascites cells in 20 mM KCl (low CKl supernatant) contained the initiation factors EIF-1 and EIF-2 (and the elongation factore EF-1 and EF-2) but lacked EIF-3; thus, low KCl supernatant could be used to assay for EIF-3. EIF-3 was prepared from a crude initiation factor perparation (a 250 mM KCl extract of ascites cell ribosomes precipitated with 70% saturated ammonium sulfate) by chromatography on DEAE-Sephadex A-50 and hydroxylapatite. The EIF-O had no detectable EIF-1 and little or no EIF-2. Factor EIF-3 was required fro translation of encephalomyocarditis virus RNA. The molecular weight of EIF-3 was estimated by Sephadex G-200 filtration to be 139,000; the sedimentation coefficient was calculated to be about 5.8. EIF-3 formed a binary complex specifically with the initiator tRNA, Met-tRNAf, and if GTP was present the factor formed a ternary complex (EIF-3-Met-tRNAf-GTP). The EIF-3 preparation had no methionyl-tRNA synthetase activity to account for binding. Complex-formation was with eukaryotic Met-tRNAf and no other aminoacyl-tRNA. The binary and ternary complexes were retained quantitatively on Millipore filters (which was the most convenient assay), but they could also be demonstrated by filtration through Sephadex G-100 or by glycerol gradient centrifugation. GTP increased the rate, the amount, and the stability of complex formed; the ration of GTP to Met-tRNAf in the ternary complex appeared to be 1. The binary and the ternary complexes transferred Met-tRNAf to the 40 S ribosomal subunits, but not to 60 S subparticles. The factor-dependent binding of Met-tRNAf to the 40 S subunit did not require mRNA (or GTP). In the presence of 60 S subunits, the initiator tRNA bound to 40 S subunits was not transferred to 80 S ribosomes even if mRNA was added--that reaction may require another initiation factor. Treatment of EIF-3 with N-ethylmaleimide led to loss of its activity in complex formation and in support of the translation of encephalomyocarditis virus RNA. In addition to forming the binary and ternary complexes, and supporting the translation of encephalomyocarditis virus RNA, EIF-3 also increases the number of free ribosomal subunits by either preventing their association or causing dissociation of 80 S couples.