Gluick T C, Draper D E
Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218.
J Mol Biol. 1994 Aug 12;241(2):246-62. doi: 10.1006/jmbi.1994.1493.
A sequence in the leader and first gene of the Escherichia coli alpha mRNA folds into a complex pseudoknot structure that is required for binding of a translational repressor. The thermal denaturation of a 112 nt RNA containing this structure has been followed by calorimetry and UV hyperchromicity. To determine the partially folded intermediates in unfolding, the denaturation of 13 mutants and of several fragments with successive deletions of helices were investigated as well. An unfolding pathway with seven states is proposed as the simplest mechanism that accounts for the data, and has several implications. (1) The lowest temperature transition appears only in the presence of moderate concentrations of Mg2+ or high concentrations of K+ (delta H approximately 45 kcal/mol), and is the unfolding of tertiary structures, rather than secondary structure. Under some conditions it is destabilized by increasing salt concentration. (2) Two of the intermediates unfolding at higher temperature must have non-canonical or tertiary interactions in addition to the known secondary structure. (3) Two alternative structures compete for formation of the complete pseudoknot, and form as the pseudoknot unfolds. Thus structures not present in the completely folded pseudoknot affect the overall thermodynamics, and probably the kinetics, of unfolding. (4) Approximately 16 kcal/mol of free energy is required to completely expose the coding region to ribosomes at 37 degrees C, though approximately 6.5 kcal/mol is regained by refolding of upstream regions after the pseudoknot is unfolded. The substantial energy needed to unfold the pseudoknot may affect the rate of translation from this ribosome binding site. A simple model of RNA folding in which an optimum secondary structure forms first, followed by tertiary interactions that further stabilize the secondary structure, does not hold in this RNA.
大肠杆菌α mRNA前导区和第一个基因中的一个序列折叠成一种复杂的假结结构，这是翻译阻遏物结合所必需的。含有该结构的112 nt RNA的热变性已通过量热法和紫外增色法进行了跟踪。为了确定解折叠过程中的部分折叠中间体，还研究了13个突变体以及几个螺旋连续缺失片段的变性情况。提出了一个具有七个状态的解折叠途径，作为解释这些数据的最简单机制，并且有几个含义。（1）最低温度转变仅在中等浓度的Mg2+或高浓度的K+存在时出现（ΔH约为45 kcal/mol），并且是三级结构的解折叠，而不是二级结构。在某些条件下，它会因盐浓度的增加而不稳定。（2）在较高温度下解折叠的两个中间体除了已知的二级结构外，还必须具有非经典或三级相互作用。（3）两种替代结构竞争形成完整的假结，并在假结解折叠时形成。因此，完全折叠的假结中不存在的结构会影响解折叠的整体热力学，可能还会影响动力学。（4）在37℃下将编码区完全暴露于核糖体需要约16 kcal/mol的自由能，尽管在假结解折叠后上游区域重新折叠可恢复约6.5 kcal/mol的能量。解折叠假结所需的大量能量可能会影响从这个核糖体结合位点进行翻译的速率。一个简单的RNA折叠模型，即首先形成最佳二级结构，然后通过三级相互作用进一步稳定二级结构，在这种RNA中并不成立。
