1] Department of Applied Physics, Stanford University, Stanford, California 94305-4090, USA [2] Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5126, USA.
Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5126, USA.
Nature. 2014 Aug 21;512(7514):328-32. doi: 10.1038/nature13428. Epub 2014 Jun 11.
Spontaneous changes in the reading frame of translation are rare (frequency of 10(-3) to 10(-4) per codon), but can be induced by specific features in the messenger RNA (mRNA). In the presence of mRNA secondary structures, a heptanucleotide 'slippery sequence' usually defined by the motif X XXY YYZ, and (in some prokaryotic cases) mRNA sequences that base pair with the 3' end of the 16S ribosomal rRNA (internal Shine-Dalgarno sequences), there is an increased probability that a specific programmed change of frame occurs, wherein the ribosome shifts one nucleotide backwards into an overlapping reading frame (-1 frame) and continues by translating a new sequence of amino acids. Despite extensive biochemical and genetic studies, there is no clear mechanistic description for frameshifting. Here we apply single-molecule fluorescence to track the compositional and conformational dynamics of individual ribosomes at each codon during translation of a frameshift-inducing mRNA from the dnaX gene in Escherichia coli. Ribosomes that frameshift into the -1 frame are characterized by a tenfold longer pause in elongation compared to non-frameshifted ribosomes, which translate through unperturbed. During the pause, interactions of the ribosome with the mRNA stimulatory elements uncouple EF-G catalysed translocation from normal ribosomal subunit reverse-rotation, leaving the ribosome in a non-canonical intersubunit rotated state with an exposed codon in the aminoacyl-tRNA site (A site). tRNA(Lys) sampling and accommodation to the empty A site and EF-G action either leads to the slippage of the tRNAs into the -1 frame or maintains the ribosome into the 0 frame. Our results provide a general mechanistic and conformational framework for -1 frameshifting, highlighting multiple kinetic branchpoints during elongation.
翻译自发的阅读框改变在翻译中很少见（每密码子的频率为 10(-3) 到 10(-4)），但可以被信使 RNA（mRNA）中的特定特征诱导。在存在 mRNA 二级结构的情况下，一个七核苷酸“滑动序列”通常由 motif X XXY YYZ 定义，并且（在一些原核情况下）与 16S 核糖体 rRNA 的 3'端碱基配对的 mRNA 序列（内部 Shine-Dalgarno 序列），特定的有计划的框架改变发生的可能性增加，其中核糖体向后移动一个核苷酸进入重叠的阅读框架（-1 框架），并通过翻译新的氨基酸序列继续。尽管进行了广泛的生化和遗传研究，但对于移框没有明确的机制描述。在这里，我们应用单分子荧光技术，在大肠杆菌的 dnaX 基因的移框诱导 mRNA 的翻译过程中，跟踪每个密码子的单个核糖体的组成和构象动力学。与非移框核糖体相比，移框到-1 框架的核糖体在延伸过程中延长的停顿时间长十倍，非移框核糖体则不受干扰地翻译。在停顿期间，核糖体与 mRNA 刺激元件的相互作用使 EF-G 催化的易位与正常核糖体亚基反向旋转解耦，使核糖体处于非典型的亚基旋转状态，氨酰-tRNA 位（A 位）暴露密码子。tRNA(Lys)的采样和适应空的 A 位以及 EF-G 的作用，要么导致 tRNAs 滑入-1 框架，要么使核糖体保持在 0 框架。我们的结果为-1 移框提供了一个通用的机制和构象框架，突出了延伸过程中的多个动力学分支点。
