Department of Chemistry and Biochemistry, the Colorado College, Colorado Springs, Colorado 80903, USA.
RNA. 2011 Apr;17(4):710-7. doi: 10.1261/rna.2263211. Epub 2011 Mar 2.
Riboswitches are functional mRNA that control gene expression. Thiamine pyrophosphate (TPP) binds to thi-box riboswitch RNA and allosterically inhibits genes that code for proteins involved in the biosynthesis and transport of thiamine. Thiamine binding to the pyrimidine sensor helix and pyrophosphate binding to the pyrophosphate sensor helix cause changes in RNA conformation that regulate gene expression. Here we examine the thermodynamic properties of the internal loop of the pyrophosphate binding domain by comparing the wild-type construct (RNA WT) with six modified 2 x 2 bulged RNA and one 2 x 2 bulged DNA. The wild-type construct retains five conserved bases of the pyrophosphate sensor domain, two of which are in the 2 x 2 bulge (C65 and G66). The RNA WT construct was among the most stable (ΔG°₃₇ = -7.7 kcal/mol) in 1 M KCl at pH 7.5. Breaking the A•G mismatch of the bulge decreases the stability of the construct ~0.5-1 kcal/mol, but does not affect magnesium binding to the RNA WT. Guanine at position 48 is important for RNA-Mg²+ interactions of the TPP-binding riboswitch at pH 7.5. In the presence of 9.5 mM magnesium at pH 5.5, the bulged RNA constructs gained an average of 1.1 kcal/mol relative to 1 M salt. Formation of a single A+•C mismatch base pair contributes about 0.5 kcal/mol at pH 5.5, whereas two tandem A+•C mismatch base pairs together contribute about 2 kcal/mol.
Riboswitches 是一种功能性的 mRNA，可以控制基因表达。焦磷酸硫胺素（TPP）与硫代盒核糖开关 RNA 结合，并变构抑制编码参与硫胺素生物合成和运输的蛋白质的基因。硫胺素与嘧啶传感器螺旋结合，焦磷酸与焦磷酸传感器螺旋结合，导致 RNA 构象发生变化，从而调节基因表达。在这里，我们通过比较野生型构建体（RNA WT）与六个修饰的 2 x 2 膨出 RNA 和一个 2 x 2 膨出 DNA，来研究焦磷酸结合结构域内部环的热力学性质。野生型构建体保留了焦磷酸传感器结构域的五个保守碱基，其中两个位于 2 x 2 膨出处（C65 和 G66）。在 pH 7.5、1 M KCl 中，RNA WT 构建体是最稳定的（ΔG°₃₇=-7.7 kcal/mol）之一。破坏膨出处的 A•G 错配会使构建体的稳定性降低约 0.5-1 kcal/mol，但不影响镁与 RNA WT 的结合。在 pH 7.5 下，位置 48 的鸟嘌呤对 TPP 结合核糖开关的 RNA-Mg²+相互作用很重要。在 pH 5.5、9.5 mM 镁存在的情况下，与 1 M 盐相比，膨出 RNA 构建体平均获得了 1.1 kcal/mol。在 pH 5.5 下，形成一个 A+•C 错配碱基对大约贡献 0.5 kcal/mol，而两个串联的 A+•C 错配碱基对一起贡献约 2 kcal/mol。
