The model mRNA (MR), 11-mer RNA containing two nitroxide spin labels at the 5′- and 3′-terminal nucleotides and prone to form a stable homodimer (MR)₂, was used for Electron Paramagnetic Resonance study of structural rearrangements in mRNA occurring upon its binding to human 80S ribosomes. The formation of two different types of ribosomal complexes with MR was observed. First, there were stable complexes where MR was fixed in the ribosomal mRNA-binding channel by the codon-anticodon interaction(s) with cognate tRNA(s). Second, we for the first time detected complexes assembled without tRNA due to the binding of MR most likely to an exposed peptide of ribosomal protein uS3 away from the mRNA channel. The analysis of interspin distances allowed the conclusion that 80S ribosomes facilitate dissociation of the duplex (MR)₂: the equilibrium between the duplex and the single-stranded MR shifts to MR due to its efficient binding with ribosomes. Furthermore, we observed a significant influence of tRNA bound at the ribosomal exit (E) and/or aminoacyl (A) sites on the stability of ribosomal complexes. Our findings showed that a part of mRNA bound in the ribosome channel, which is not involved in codon-anticodon interactions, has more degrees of freedom than that interacting with tRNAs.

中文翻译：

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通过EPR光谱揭示与人80S核糖体结合后mRNA的结构重排

模型mRNA（MR），11-mer RNA，在5'-和3'-末端核苷酸处含有两个氮氧自由基自旋标记，易于形成稳定的同型二聚体（MR）₂被用于电子顺磁共振研究与人80S核糖体结合后发生的mRNA结构重排。观察到两种不同类型的核糖体复合物与MR的形成。首先，存在稳定的复合物，其中MR通过与同源tRNA的密码子-反密码子相互作用而固定在核糖体mRNA结合通道中。其次，由于MR极有可能与核糖体蛋白uS3的暴露肽远离mRNA通道的结合，我们首次检测到没有tRNA组装的复合物。旋转距离的分析可以得出这样的结论，即80S核糖体有助于双链体（MR）_2的解离：双链体和单链MR之间的平衡因其与核糖体的有效结合而转变为MR。此外，我们观察到在核糖体出口（E）和/或氨酰基（A）位点上结合的tRNA对核糖体复合物稳定性的显着影响。我们的发现表明，与核糖体通道结合的不参与密码子-反密码子相互作用的部分mRNA比与tRNA相互作用的自由度更高。