PDF(Pubmed)
Abstract：
The two main steps of translation, peptidyl transfer, and translocation are accompanied by counterclockwise and clockwise rotations of the large and small ribosomal subunits with respect to each other. Upon peptidyl transfer, the small ribosomal subunit rotates counterclockwise relative to the large subunit, placing the ribosome into the rotated conformation. Simultaneously, tRNAs move into the hybrid conformation, and the L1 stalk moves inward toward the P-site tRNA. The conformational dynamics of pretranslocation ribosomes were extensively studied by ensemble and single-molecule methods. Different experimental modalities tracking ribosomal subunits, tRNAs, and the L1 stalk showed that pretranslocation ribosomes undergo spontaneous conformational transitions. Thus, peptidyl transfer unlocks the ribosome and decreases an energy barrier for the reverse ribosome rotation during translocation. However, the tracking of translation with ribosomes labeled at rRNA helices h44 and H101 showed a lack of spontaneous rotations in pretranslocation complexes. Therefore, reverse intersubunit rotations occur during EF-G catalyzed translocation. To reconcile these views, we used high-speed single-molecule microscopy to follow translation in real time. We showed spontaneous rotations in puromycin-released h44-H101 dye-labeled ribosomes. During elongation, the h44-H101 ribosomes undergo partial spontaneous rotations. Spontaneous rotations in h44-H101-labeled ribosomes are restricted prior to aminoacyl-tRNA binding. The pretranslocation h44-H101 ribosomes spontaneously exchanged between three different rotational states. This demonstrates that peptidyl transfer unlocks spontaneous rotations and pretranslocation ribosomes can adopt several thermally accessible conformations, thus supporting the Brownian model of translocation.
摘要：
翻译的两个主要步骤，肽基转移，和易位伴随着大核糖体亚基和小核糖体亚基相对于彼此的逆时针和顺时针旋转。肽基转移后，小核糖体亚基相对于大亚基逆时针旋转，将核糖体置于旋转的构象中。同时,tRNAs进入杂交构象，L1茎向内向P位点tRNA移动。通过集成和单分子方法广泛研究了易位核糖体的构象动力学。追踪核糖体亚基的不同实验模式，tRNAs,L1茎显示转位前核糖体经历自发的构象转变。因此，肽基转移解锁核糖体并降低易位过程中核糖体反向旋转的能障。然而,在rRNA螺旋h44和H101标记的核糖体的翻译跟踪显示，在转位前复合物中缺乏自发旋转。因此,在EF-G催化的易位过程中发生反向亚基间旋转。为了调和这些观点，我们使用高速单分子显微镜实时跟踪翻译。我们显示了嘌呤霉素释放的h44-H101染料标记的核糖体的自发旋转。在伸长期间,h44-H101核糖体经历部分自发旋转。在氨酰基-tRNA结合之前，h44-H101标记的核糖体的自发旋转受到限制。前易位h44-H101核糖体在三种不同的旋转状态之间自发交换。这表明肽基转移解锁了自发旋转和转位前核糖体可以采用几种可热接近的构象，从而支持布朗模型的易位。
