Molecular dissection of the silkworm ribosomal stalk complex: the role of multiple copies of the stalk proteins.
Molecular dissection of the silkworm ribosomal stalk complex: the role of multiple copies of the stalk proteins.
复制标题
蚕核糖体茎复合物的分子解剖：茎蛋白的多个副本的作用。
DOI：
10.1093/nar/gkt044
复制
发表时间：
2013-04-01
影响因子：
14.9
通讯作者：
Uchiumi T
中科院分区：
文献类型：
--
作者：
Baba K;Tumuraya K;Tanaka I;Yao M;Uchiumi T
在动物核糖体中，两个茎蛋白P1和P2形成一个异二聚体，这两个二聚体与锚蛋白P0组成一个五聚体复合体，对翻译GTP酶因子向核糖体募集至关重要。为了研究茎蛋白每个拷贝的功能贡献，我们构建了P0突变体，其中两个C-末端螺旋之一，即螺旋I(N-末端侧)或螺旋II(C-末端一侧)不能与P1-P2二聚体结合。我们还构建了由完整的P1/P2单体和一个CTD-截短的配对组成的‘一个C-末端结构域茎二聚体’，即P1-P2ΔC和P1ΔC-P2。通过P0和P1-P2变异体的组合，重组了不同的复合体，并在依赖EEF-2的GTP酶和依赖EEF-1的α/EEF-2依赖的聚苯丙氨酸的体外合成实验中测试了它们的功能。与螺旋I结合的双/单CTD二聚体的活性高于与螺旋II结合的双/单CTD二聚体。尽管单一CTD二聚体的多肽合成活性较低，但其与两个螺旋的结合活性显著提高，表明两个茎二聚体相互合作，特别是在多肽合成过程中。连接P0两个螺旋的保守YPT序列发生突变后，两个茎二聚体对活性的促进作用就消失了，这表明该序列在两个茎二聚体的协同作用中发挥了作用。
In animal ribosomes, two stalk proteins P1 and P2 form a heterodimer, and the two dimers, with the anchor protein P0, constitute a pentameric complex crucial for recruitment of translational GTPase factors to the ribosome. To investigate the functional contribution of each copy of the stalk proteins, we constructed P0 mutants, in which one of the two C-terminal helices, namely helix I (N-terminal side) or helix II (C-terminal side) were unable to bind the P1–P2 dimer. We also constructed ‘one-C-terminal domain (CTD) stalk dimers’, P1–P2ΔC and P1ΔC–P2, composed of intact P1/P2 monomer and a CTD-truncated partner. Through combinations of P0 and P1–P2 variants, various complexes were reconstituted and their function tested in eEF-2-dependent GTPase and eEF-1α/eEF-2-dependent polyphenylalanine synthesis assays in vitro. Double/single-CTD dimers bound to helix I showed higher activity than that bound to helix II. Despite low polypeptide synthetic activity by a single one-CTD dimer, its binding to both helices considerably increased activity, suggesting that two stalk dimers cooperate, particularly in polypeptide synthesis. This promotion of activity by two stalk dimers was lost upon mutation of the conserved YPT sequence connecting the two helices of P0, suggesting a role for this sequence in cooperativity of two stalk dimers.