The indispensable N-terminal half of eIF3j/HCR1 cooperates with its structurally conserved binding partner eIF3b/PRT1-RRM and with eIF1A in stringent AUG selection.
The indispensable N-terminal half of eIF3j/HCR1 cooperates with its structurally conserved binding partner eIF3b/PRT1-RRM and with eIF1A in stringent AUG selection.
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DOI：
10.1016/j.jmb.2009.12.047
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发表时间：
2010-03-05
影响因子：
5.6
通讯作者：
Valásek L
中科院分区：
文献类型：
--
作者：
Elantak L;Wagner S;Herrmannová A;Karásková M;Rutkai E;Lukavsky PJ;Valásek L
尽管近年来我们对真核生物翻译起始因子3 （eIF3）单个亚基的许多功能的了解取得了进展，但在分子水平上仍然知之甚少。利用核磁共振光谱，我们确定了eIF3亚基之间相互作用的第一个溶液结构。我们发现，人类eIF3j n端酸性结构域（NTA）中的一个保守色氨酸残基位于人类eIF3b-RRM的螺旋α1 -环L5疏水口袋中。突变其酵母同源物中相应的“口袋”残基会降低细胞生长速度，在体外和体内消除eIF3j/HCR1与eIF3b/PRT1的关联，影响eIF3的40s占用，并产生泄漏扫描缺陷，表明AUG选择过程的放松。出乎意料的是，我们发现含有NTA基序的eIF3j/HCR1的n端一半（NTD）对于酵母细胞的野生型生长是必不可少的和足够的。此外，我们证明，j/HCR1或其NTD的缺失，或关键色氨酸残基的突变，都会导致严重的泄漏扫描表型，部分被过度表达的eIF1A抑制，这被认为是在正确的起始密码子处稳定正确形成的起始前复合物。这些发现表明eIF3j/HCR1仍然与扫描起始前复合物相关，并且在mRNA募集时不会像之前认为的那样与小核糖体亚基分离。最后，我们通过鉴定eIF3j/HCR1与mRNA进入通道附近的小核糖体蛋白RPS2和RPS23的特异性相互作用，为早期绘制人类eIF3j核糖体结合位点提供了进一步的支持。综上所述，我们提出eIF3j/HCR1与eIF3b/PRT1-RRM和eIF1A在核糖体上密切合作，以确保正确形成严格的AUG识别所需的扫描阻止构象。
Despite the recent progress in our understanding of the numerous functions of individual subunits of eukaryotic translation initiation factor 3 (eIF3), there is still only little known on the molecular level. Using NMR spectroscopy, we determined the first solution structure of an interaction between eIF3 subunits. We revealed that a conserved tryptophan residue in the human eIF3j N-terminal acidic domain (NTA) is held in the helix α1 – loop L5 hydrophobic pocket of the human eIF3b-RRM. Mutating the corresponding “pocket” residues in its yeast orthologue reduces cellular growth rate, eliminates eIF3j/HCR1 association with eIF3b/PRT1 in vitro and in vivo, affects 40S-occupancy of eIF3, and produces a leaky scanning defect indicative of a deregulation of the AUG selection process. Unexpectedly, we found that the N-terminal half (NTD) of eIF3j/HCR1 containing the NTA motif is indispensable and sufficient for wild-type growth of yeast cells. Furthermore, we demonstrate that deletion of either j/HCR1 or its NTD only, or mutating the key tryptophan residues results in the severe leaky scanning phenotype partially suppressible by overexpressed eIF1A, which is thought to stabilize properly formed pre-initiation complexes at the correct start codon. These findings indicate that eIF3j/HCR1 remains associated with the scanning pre-initiation complexes and does not dissociate from the small ribosomal subunit upon mRNA recruitment as previously believed. Finally, we provide further support for earlier mapping of the ribosomal binding site for human eIF3j by identifying specific interactions of eIF3j/HCR1 with small ribosomal proteins RPS2 and RPS23 located in the vicinity of the mRNA entry channel. Taken together we propose that eIF3j/HCR1 closely co-operates with eIF3b/PRT1-RRM and eIF1A on the ribosome to ensure proper formation of the scanning-arrested conformation required for stringent AUG recognition.
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