Jacobson A B, Arora R, Zuker M, Priano C, Lin C H, Mills D R
Department of Microbiology, State University of New York, Stony Brook 11794-5222, USA.
J Mol Biol. 1998 Jan 30;275(4):589-600. doi: 10.1006/jmbi.1997.1472.
We have analyzed both conformational and functional changes caused by two large cis-acting deletions (delta 159 and delta 549) located within the read-through domain, a 850 nucleotide hairpin, in coliphage Q beta genomic RNA. Studies in vivo show that co-translational regulation of the viral coat and replicase genes has been uncoupled in viral genomes carrying deletion delta 159. Translational regulation is restored in deletion delta 549, a naturally evolved pseudorevertant. Structural analysis by computer modeling shows that structural features within the read-through domain of delta 159 RNA are less well determined than they are in the read-through domain of wild-type RNA, whereas predicted structure in the read-through domain of evolved pseudorevertant delta 549 is unusually well determined. Structural analysis by electron microscopy of the genomic RNAs shows that several long range helices at the base of the read-through domain, that suppress translational initiation of the viral replicase gene in the wild-type genome, have been destabilized in delta 159 RNA. In addition, the structure of local hairpins within the read-through region is more variable in delta 159 RNA than in wild-type RNA. Stable RNA secondary structure is restored in the read-through domain of delta 549 RNA. Our analyses suggest that structure throughout the read-through domain affects the regulation of viral replicase expression by altering the likelihood that long-range interactions at the base of the domain will form. We discuss possible kinetic and equilibrium models that can explain this effect, and argue that observed changes in structural plasticity within the read-through domain of the mutant genomes are key in understanding the process. During the course of these studies, we became aware of the importance of the information contained in the energy dot plot produced by the RNA secondary structure prediction program mfold. As a result, we have improved the graphical representation of this information through the use of color annotation in the predicted optimal folding. The method is presented here for the first time.
我们分析了位于噬菌体Qβ基因组RNA通读域（一个850个核苷酸的发夹结构）内的两个大的顺式作用缺失（δ159和δ549）所引起的构象和功能变化。体内研究表明，在携带缺失δ159的病毒基因组中，病毒外壳蛋白和复制酶基因的共翻译调控已解偶联。在天然进化的假回复突变体缺失δ549中，翻译调控得以恢复。通过计算机建模进行的结构分析表明，δ159 RNA通读域内的结构特征不如野生型RNA通读域中的结构特征确定得好，而进化后的假回复突变体δ549通读域中的预测结构确定得异常好。通过电子显微镜对基因组RNA进行的结构分析表明，通读域底部的几个长程螺旋在野生型基因组中抑制病毒复制酶基因的翻译起始，在δ159 RNA中已不稳定。此外，通读区域内局部发夹的结构在δ159 RNA中比在野生型RNA中更具变异性。δ549 RNA的通读域中恢复了稳定的RNA二级结构。我们的分析表明，通读域内的结构通过改变该区域底部长程相互作用形成的可能性来影响病毒复制酶表达的调控。我们讨论了可以解释这种效应的可能的动力学和平衡模型，并认为在突变基因组通读域内观察到的结构可塑性变化是理解这一过程的关键。在这些研究过程中，我们意识到RNA二级结构预测程序mfold产生的能量点图中所包含信息的重要性。因此，我们通过在预测的最佳折叠中使用颜色注释改进了该信息的图形表示。该方法首次在此处提出。
