Hüttenhofer A, Westhof E, Böck A
Lehrstuhl für Mikrobiologie der Universität München, Germany.
RNA. 1996 Apr;2(4):354-66.
On the basis of chemical probing data, the solution structures of RNA hairpins within fdhF and fdnG mRNAs in Escherichia coli, which both promote selenocysteine incorporation at UGA codons, were derived with the help of computer modeling. We find that these mRNA hairpins contain two separate structural domains that possibly also exert two different functions. The first domain is comprised of the UGA codon, which is included within a complex and distorted double-stranded region. Thereby, release factor 2 might be prevented from binding to the UGA codon to terminate protein synthesis. The second domain is located within the apical loop of the mRNA hairpin structures. This loop region exhibits a defined tertiary structure in which no base is involved in Watson-Crick interactions. The structure of the loop is such that, following a sharp turn after G22 (A22 in fdnG mRNA), bases G23 and U24 are exposed to the solvent on the deep groove side of the supporting helix. Residues C25 and U26 close the loop with a possible single H-bonding interaction between the first and last residues of the loop, 04(U26) and N6(A21). The bulge residues U17 and U18 (in fdhF mRNA), or Ul7 only in fdnG mRNA, point their Watson-Crick positions in the same direction as loop residues G23 and U24 do, and at the same time open up the deep groove at the top of the hairpin helix. Chemical probing data demonstrate that bases G23 and U24 in both mRNA hairpins, as well as residues U17 and Ul7/U18 (for fdhF mRNA) located in a bulge 5' to the loop, are involved directly in binding to special elongation factor SELB in both mRNAs. Therefore, SELB recognizes identical bases within both mRNA hairpins despite differences in their primary sequence, consistent with the derived 3D models for these mRNAs, which exhibit similar tertiary structures. Binding of SELB to the fdhF mRNA hairpin was estimated to proceed with an apparent Kd of 30 nM.
基于化学探测数据，借助计算机建模得出了大肠杆菌中fdhF和fdnG mRNA内RNA发夹的溶液结构，这两种发夹均能促进UGA密码子处硒代半胱氨酸的掺入。我们发现这些mRNA发夹包含两个独立的结构域，可能也发挥两种不同的功能。第一个结构域由UGA密码子组成，它包含在一个复杂且扭曲的双链区域内。由此，可能会阻止释放因子2与UGA密码子结合以终止蛋白质合成。第二个结构域位于mRNA发夹结构的顶端环内。该环区域呈现出一种特定的三级结构，其中没有碱基参与沃森-克里克相互作用。环的结构使得在G22（fdnG mRNA中的A22）之后急剧转弯后，碱基G23和U24在支撑螺旋的深沟侧暴露于溶剂中。残基C25和U26封闭环，环的第一个和最后一个残基04（U26）和N6（A21）之间可能存在单一氢键相互作用。凸起残基U17和U18（在fdhF mRNA中），或仅在fdnG mRNA中的U17，其沃森-克里克位置与环残基G23和U24的方向相同，同时在发夹螺旋顶部打开深沟。化学探测数据表明，两种mRNA发夹中的碱基G23和U24，以及位于环5'端凸起处的残基U17和U17 / U18（对于fdhF mRNA），都直接参与与两种mRNA中特殊延伸因子SELB的结合。因此，尽管两种mRNA发夹的一级序列存在差异，但SELB识别它们内部相同的碱基，这与这些mRNA的三维模型一致，这些模型呈现出相似的三级结构。估计SELB与fdhF mRNA发夹的结合表观解离常数Kd为30 nM。
