Single-nucleotide control of tRNA folding cooperativity under near-cellular conditions
Single-nucleotide control of tRNA folding cooperativity under near-cellular conditions
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DOI：
10.1073/pnas.1913418116
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发表时间：
2019-11-12
影响因子：
11.1
通讯作者：
Bevilacqua, Philip C.
中科院分区：
文献类型：
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作者：
Leamy, Kathleen A.;Yamagami, Ryota;Bevilacqua, Philip C.
RNA折叠通常是通过体外再生全长RNA和跟踪折叠转变来研究的。然而，当细胞内转录物从RNA聚合酶出现时，它会折叠。在这里，我们研究了酵母和大肠杆菌转移rna （tRNAs）的许多转录后期中间体的折叠途径和稳定性。转移RNA是一种高度调控的功能性RNA，它经历了转录后加工的多个步骤，并且在细胞内的生命周期中具有非常不同的长度。前体转录本在三叶草核心的5‘和3’端延伸，这些延伸在添加3'-CCA和氨基酰化之前被修剪。我们研究了前体tRNA和三叶草结构的转录后期中间体的热力学和结构。我们使用多种生化和生物物理方法在二级和三级结构水平上检查RNA折叠。我们的研究结果表明，也许大自然选择了单碱基来控制功能性3D结构的折叠。在近细胞条件下，酵母tRNA（Phe）和大肠杆菌tRNA（Ala）转录物只有在三叶草中几乎所有核苷酸都通过间接增强折叠协同性被转录时才能以单一的合作转变进行折叠。此外，5‘和3’端上的原生扩展不会干扰核心的协同折叠。这种高度控制的协同折叠对细胞中tRNA的加工和修饰酶的识别以及tRNA的质量控制具有重要意义。
RNA folding is often studied by renaturing full-length RNA in vitro and tracking folding transitions. However, the intracellular transcript folds as it emerges from the RNA polymerase. Here, we investigate the folding pathways and stability of numerous late-transcriptional intermediates of yeast and Escherichia coli transfer RNAs (tRNAs). Transfer RNA is a highly regulated functional RNA that undergoes multiple steps of posttranscriptional processing and is found in very different lengths during its lifetime in the cell. The precursor transcript is extended on both the 5' and 3' ends of the cloverleaf core, and these extensions get trimmed before addition of the 3'-CCA and aminoacylation. We studied the thermodynamics and structures of the precursor tRNA and of late-transcriptional intermediates of the cloverleaf structure. We examined RNA folding at both the secondary and tertiary structural levels using multiple biochemical and biophysical approaches. Our findings suggest that perhaps nature has selected for a single-base addition to control folding to the functional 3D structure. In near-cellular conditions, yeast tRNA(Phe) and E. coli tRNA(Ala) transcripts fold in a single, cooperative transition only when nearly all of the nucleotides in the cloverleaf are transcribed by indirectly enhancing folding cooperativity. Furthermore, native extensions on the 5' and 3' ends do not interfere with cooperative core folding. This highly controlled cooperative folding has implications for recognition of tRNA by processing and modification enzymes and quality control of tRNA in cells.