Mirmira S R, Tinoco I
Department of Chemistry, University of California, Berkeley, USA.
Biochemistry. 1996 Jun 18;35(24):7675-83. doi: 10.1021/bi960415q.
The solution structure of a 16-nucleotide RNA hairpin, 5'-GCCUAG[CAAC]CUGGGC (loop bases in square brackets), has been determined by proton, phosphorus, and carbon (natural abundance) nuclear magnetic resonance (NMR) spectroscopy. This RNA tetraloop hairpin varies in four loop nucleotides from the wild-type T4 RNA hairpin (with eight loop nucleotides) involved in the translational repression of bacteriophage T4 DNA polymerase. Despite the differences in their sequence and proposed secondary structures, these two hairpins bind T4 DNA polymerase with equal affinity. The NMR spectra of the mutant hairpin indicate that its stem is extended in comparison to that of the wild-type hairpin by the formation of two additional Watson-Crick base pairs. The NMR data provide a precisely defined structure for the mutant hairpin with an average root mean square deviation of approximately 0.7 A for all 16 residues in the molecule. The structure of the mutant loop is very similar to that determined previously for the wild-type hairpin. The three loop bases that are conserved between the mutant and wild-type hairpins point out in solution with the groups capable of hydrogen bond formation exposed to the solution. This is exactly what was seen for the wild-type hairpin. Also, unusual, long-range NOEs, loop hydrogen bonds, and even the position at which the loop bends are common features between the two loops. This explains how two different hairpins, by adopting similar three-dimensional structures, have the same affinity for the DNA polymerase.
通过质子、磷和碳（天然丰度）核磁共振（NMR）光谱法，已确定了一个16核苷酸RNA发夹结构，即5'-GCCUAG[CAAC]CUGGGC（方括号内为环核苷酸）。这个RNA四环发夹在四个环核苷酸上与参与噬菌体T4 DNA聚合酶翻译抑制的野生型T4 RNA发夹（有八个环核苷酸）不同。尽管它们的序列和推测的二级结构存在差异，但这两个发夹以相同的亲和力结合T4 DNA聚合酶。突变发夹的NMR光谱表明，与野生型发夹相比，其茎通过形成另外两个沃森-克里克碱基对而得到延伸。NMR数据为突变发夹提供了一个精确确定的结构，分子中所有16个残基的平均均方根偏差约为0.7埃。突变环的结构与先前确定的野生型发夹结构非常相似。突变型和野生型发夹之间保守的三个环碱基在溶液中向外突出，能够形成氢键的基团暴露于溶液中。这与野生型发夹的情况完全相同。此外，可以形成氢键的远距离核Overhauser效应（NOE）、环氢键，甚至环弯曲的位置，都是两个环的共同特征。这就解释了两个不同的发夹如何通过采用相似的三维结构，对DNA聚合酶具有相同的亲和力。
