Structural integrity of {alpha}-helix H12 in translation initiation factor eIF5B is critical for 80S complex stability.
Structural integrity of {alpha}-helix H12 in translation initiation factor eIF5B is critical for 80S complex stability.
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DOI：
10.1261/rna.2412511
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发表时间：
2011-04
期刊：
影响因子：
4.5
通讯作者：
B. Shin;M. Acker;Joo-Ran Kim;Kathryn N. Maher;S. Arefin;J. Lorsch;T. Dever
中科院分区：
文献类型：
--
作者：
B. Shin;M. Acker;Joo-Ran Kim;Kathryn N. Maher;S. Arefin;J. Lorsch;T. Dever
翻译起始因子eIF5B促进依赖GTP的核糖体亚基参与翻译起始途径的最后一步。该蛋白类似于一个圣杯，α-螺旋H12形成了连接GTP结合结构域杯和结构域IV碱基的茎。Helix H12被认为是一个刚性的杠杆臂，控制着结构域IV的核苷酸结合，并作为一种分子尺子固定结构域IV和因子的G结构域之间的距离。为了研究其功能，将螺旋H12延长或缩短一到两圈。此外，通过用甘氨酸取代螺旋上的6个连续残基来改变螺旋的刚性。而突变对因子与核糖体的结合及其GTP结合和水解活性的影响很小，将螺旋缩短6个残基会降低亚基在体外的连接率，该突变和Gly取代突变都会降低Met-tRNA(I)(Met)与80S络合物结合的产量。因此，这两个损害酵母细胞生长和增强体内核糖体泄漏扫描的突变，损害了亚基连接的80s产物的形成速度和稳定性。这些数据支持这样的观点，即螺旋H12作为一把尺子将核糖体的GTPase中心连接到Met-tRNA(I)(Met)结合的P位，并且螺旋H12的刚性是稳定Met-tRNA(I)(Met)结合所必需的。
Translation initiation factor eIF5B promotes GTP-dependent ribosomal subunit joining in the final step of the translation initiation pathway. The protein resembles a chalice with the α-helix H12 forming the stem connecting the GTP-binding domain cup to the domain IV base. Helix H12 has been proposed to function as a rigid lever arm governing domain IV movements in response to nucleotide binding and as a molecular ruler fixing the distance between domain IV and the G domain of the factor. To investigate its function, helix H12 was lengthened or shortened by one or two turns. In addition, six consecutive residues in the helix were substituted by Gly to alter the helical rigidity. Whereas the mutations had minimal impacts on the factor's binding to the ribosome and its GTP binding and hydrolysis activities, shortening the helix by six residues impaired the rate of subunit joining in vitro and both this mutation and the Gly substitution mutation lowered the yield of Met-tRNA(i)(Met) bound to 80S complexes formed in the presence of nonhydrolyzable GTP. Thus, these two mutations, which impair yeast cell growth and enhance ribosome leaky scanning in vivo, impair the rate of formation and stability of the 80S product of subunit joining. These data support the notion that helix H12 functions as a ruler connecting the GTPase center of the ribosome to the P site where Met-tRNA(i)(Met) is bound and that helix H12 rigidity is required to stabilize Met-tRNA(i)(Met) binding.