Costantino David, Kieft Jeffrey S
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Mail stop 8101, P.O. Box 6511, Aurora, CO 80045, USA.
RNA. 2005 Mar;11(3):332-43. doi: 10.1261/rna.7184705.
The internal ribosome site RNA of the cricket paralysis-like viruses (CrPV-like) binds directly to the ribosome, assembling the translation machinery without initiation factors. This mechanism does not require initiator tRNA, and translation starts from a non-AUG codon. A wealth of biochemical data has yielded a working model for this process, but the three-dimensional structure and biophysical characteristics of the unbound CrPV-like IRES RNAs are largely unexplored. Here, we demonstrate that the CrPV-like IRESes prefold into a two-part structure in the presence of magnesium ions. The largest part is a prefolded compact RNA domain that shares folding and structural characteristics with other compactly folded RNAs such as group I intron RNAs and RNase P RNA. Chemical probing reveals that the CrPV-like IRES' compact domain contains RNA helices that are packed tightly enough to exclude solvent, and analytical ultracentrifugation indicates a large change in the shape of the IRES upon folding. Formation of this compact domain is necessary for binding of the 40S subunit, and the structural organization of the unbound IRES RNA is consistent with the hypothesis that the IRES is functionally and structurally preorganized before ribosome binding.
蟋蟀麻痹样病毒（CrPV样）的内部核糖体位点RNA直接与核糖体结合，在没有起始因子的情况下组装翻译机器。这种机制不需要起始tRNA，翻译从非AUG密码子开始。大量的生化数据已经产生了这个过程的工作模型，但未结合的CrPV样IRES RNA的三维结构和生物物理特性在很大程度上尚未被探索。在这里，我们证明CrPV样IRES在镁离子存在下预折叠成两部分结构。最大的部分是一个预折叠的紧密RNA结构域，它与其他紧密折叠的RNA如I组内含子RNA和核糖核酸酶P RNA具有折叠和结构特征。化学探针显示，CrPV样IRES的紧密结构域包含紧密堆积以排除溶剂的RNA螺旋，分析超速离心表明IRES折叠时形状发生了很大变化。这个紧密结构域的形成对于40S亚基的结合是必要的，未结合的IRES RNA的结构组织与IRES在核糖体结合之前在功能和结构上预先组织的假设一致。
