Downs W D, Cech T R
Department of Molecular, Cellular and Developmental Biology, Howard Hughes Medical Institute, University of Colorado, Boulder 80309-0215, USA.
RNA. 1996 Jul;2(7):718-32.
The kinetics of RNA folding were examined in the L-21 ribozyme, an RNA enzyme derived from the self-splicing Tetrahymena intron. Three UV-inducible crosslinks were mapped, characterized, and used as indicators for the folded state of the ribozyme. Together these data suggest that final structures are adopted first by the P4-P6 independently folding domain and only later in a region that positions the P1 helix (including the 5' splice site), a region whose folding is linked to that of a portion of the catalytic core. At intermediate times, a non-native structure forms in the region of the triple helical scaffold, which connects the major folding domains. At 30 degrees C, the unfolded ribozyme passes through these stages with a half-life of 2 min from the time magnesium cations are provided. At higher temperatures, the half-life is shortened but the order of events is unchanged. Thermal melting of the fully folded ribozyme also revealed a multi-stage process in which the steps of folding are reversed: the kinetically slowest structure is the least stable and melts first. These structures of the ribozyme also bind Mg2+ cooperatively and their relative affinity for binding seems to be a major determinant in the order of events during folding. Na+ can also substitute for Mg2+ to give rise to the same crosslinkable structures, but only at much higher concentrations. Specific binding sites for Mg2+ may make this cation particularly efficient at electrostatic stabilization during folding of these ribozyme structures.
在源自自我剪接的嗜热四膜虫内含子的L-21核酶中研究了RNA折叠动力学。绘制、表征了三个紫外线诱导的交联，并将其用作核酶折叠状态的指标。这些数据共同表明，最终结构首先由P4-P6独立折叠结构域形成，随后才在定位P1螺旋（包括5'剪接位点）的区域形成，该区域的折叠与催化核心的一部分相关联。在中间阶段，在连接主要折叠结构域的三螺旋支架区域形成非天然结构。在30摄氏度时，从提供镁阳离子开始，未折叠的核酶以2分钟的半衰期经历这些阶段。在较高温度下，半衰期缩短，但事件顺序不变。完全折叠的核酶的热解链也揭示了一个多阶段过程，其中折叠步骤是相反的：动力学上最慢的结构最不稳定，最先解链。这些核酶结构也协同结合Mg2+，它们相对的结合亲和力似乎是折叠过程中事件顺序的主要决定因素。Na+也可以替代Mg2+产生相同的可交联结构，但仅在高得多的浓度下。Mg2+的特异性结合位点可能使这种阳离子在这些核酶结构折叠过程中的静电稳定方面特别有效。
