Su Linhui Julie, Brenowitz Michael, Pyle Anna Marie
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
J Mol Biol. 2003 Dec 5;334(4):639-52. doi: 10.1016/j.jmb.2003.09.071.
Despite a growing literature on the folding of RNA, our understanding of tertiary folding in large RNAs derives from studies on a small set of molecular examples, with primary focus on group I introns and RNase P RNA. To broaden the scope of RNA folding models and to better understand group II intron function, we have examined the tertiary folding of a ribozyme (D135) that is derived from the self-splicing ai5gamma intron from yeast mitochondria. The D135 ribozyme folds homogeneously and cooperatively into a compact, well-defined tertiary structure that includes all regions critical for active-site organization and substrate recognition. When D135 was treated with increasing concentrations of Mg(2+) and then subjected to hydroxyl radical footprinting, similar Mg(2+) dependencies were seen for internalization of all regions of the molecule, suggesting a highly cooperative folding behavior. In this work, we show that global folding and compaction of the molecule have the same magnesium dependence as the local folding previously observed. Furthermore, urea denaturation studies indicate highly cooperative unfolding of the ribozyme that is governed by thermodynamic parameters similar to those for forward folding. In fact, D135 folds homogeneously and cooperatively from the unfolded state to its native, active structure, thereby demonstrating functional reversibility in RNA folding. Taken together, the data are consistent with two-state folding of the D135 ribozyme, which is surprising given the size and multi-domain structure of the RNA. The findings establish that the accumulation of stable intermediates prior to formation of the native state is not a universal feature of RNA folding and that there is an alternative paradigm in which the folding landscape is relatively smooth, lacking rugged features that obstruct folding to the native state.
尽管关于RNA折叠的文献不断增加，但我们对大型RNA三级折叠的理解源于对一小部分分子实例的研究，主要集中在I组内含子和核糖核酸酶P RNA上。为了拓宽RNA折叠模型的范围并更好地理解II组内含子的功能，我们研究了一种核酶（D135）的三级折叠，该核酶源自酵母线粒体的自我剪接ai5γ内含子。D135核酶均匀且协同地折叠成紧凑、明确的三级结构，该结构包含对活性位点组织和底物识别至关重要的所有区域。当用浓度不断增加的Mg(2+)处理D135，然后进行羟基自由基足迹分析时，分子所有区域的内化表现出相似的Mg(2+)依赖性，表明其具有高度协同的折叠行为。在这项工作中，我们表明分子的整体折叠和压缩与先前观察到的局部折叠具有相同的镁依赖性。此外，尿素变性研究表明核酶的解折叠具有高度协同性，其受与正向折叠相似的热力学参数控制。事实上，D135从未折叠状态均匀且协同地折叠成其天然的活性结构，从而证明了RNA折叠中的功能可逆性。综上所述，这些数据与D135核酶的两态折叠一致，鉴于RNA的大小和多结构域结构，这一结果令人惊讶。这些发现表明，在天然状态形成之前稳定中间体的积累并非RNA折叠的普遍特征，并且存在一种替代范式，其中折叠景观相对平滑，缺乏阻碍折叠成天然状态的崎岖特征。
