Kim C H, Tinoco I
Department of Chemistry, University of California Berkeley, CA, 94720-1460, USA.
J Mol Biol. 2001 Mar 30;307(3):827-39. doi: 10.1006/jmbi.2001.4497.
The 3'-end region of the genomic RNA of brome mosaic virus forms a tRNA-like structure that is critical for its replication. Previous studies have shown that in this region, a stem-loop structure, called SLC, is necessary and sufficient for the binding of the RNA replicase, and for RNA replication. Recently, we determined the high-resolution NMR structure of SLC, which demonstrated that a 5'-AUA-3' triloop region is an important structural element for the enzymatic recognition. We proposed that the 5'-adenine of the triloop, which is rigidly fixed ("clamped") to the stem, is a key recognition element for the replicase. To elucidate the role of this "clamped base motif" for the enzymatic recognition, we have now investigated the solution conformations of several stem-loop molecules with mutant triloops, 5'-UUA-3', 5'-GUA-3', 5'-CUA-3' and 5'-UUU-3', that destroy the enzymatic recognition. For the GUA and UUA mutants, we have obtained high-resolution solution structures using 2D NMR. All four mutants have very similar thermodynamic stabilities, and all have the same secondary structures, a triloop with a five base-paired stem helix. In addition, they have quite similar sugar puckering patterns in the triloop region. The NMR structures of the GUA and UUA show that the 5' nucleotide of the triloop (G6 in GUA or U6 in UUA) lacks the strong interactions that hold its base in a fixed position. In particular, the U6 of UUA is found in two different conformations. Neither of these two mutants has the clamped base motif that was observed in the wild-type. While UUA also shows global change in the overall triloop conformation, GUA shows a very similar triloop conformation to the wild-type except for the lack of this motif. The absence of the clamped base motif is the only common structural difference between these two mutants and the wild-type. These results clearly indicate that the loss of function of the UUA and GUA mutants comes mainly from the destruction of a small key recognition motif rather than from global changes in their triloop conformations. Based on this study, we conclude that the key structural motif in the triloop recognized by the replicase is a solution-exposed, 5'-adenine base in the triloop that is clamped to the stem helix, which is called a clamped adenine motif.
雀麦花叶病毒基因组RNA的3'末端区域形成了一种类似tRNA的结构，这对其复制至关重要。先前的研究表明，在该区域，一个名为SLC的茎环结构对于RNA复制酶的结合以及RNA复制而言是必要且充分的。最近，我们确定了SLC的高分辨率核磁共振结构，结果表明一个5'-AUA-3'三环区域是酶促识别的重要结构元件。我们提出，三环的5'-腺嘌呤牢固地固定（“夹住”）在茎上，是复制酶的关键识别元件。为了阐明这种“夹住碱基基序”在酶促识别中的作用，我们现在研究了几种带有破坏酶促识别的突变三环（5'-UUA-3'、5'-GUA-3'、5'-CUA-3'和5'-UUU-3'）的茎环分子的溶液构象。对于GUA和UUA突变体，我们使用二维核磁共振获得了高分辨率溶液结构。所有四个突变体具有非常相似的热力学稳定性，并且都具有相同的二级结构，即带有一个五碱基对茎螺旋的三环。此外，它们在三环区域具有相当相似的糖环构象模式。GUA和UUA的核磁共振结构表明，三环的5'核苷酸（GUA中的G6或UUA中的U6）缺乏将其碱基固定在固定位置的强相互作用。特别是，UUA的U6存在于两种不同的构象中。这两个突变体都没有在野生型中观察到的夹住碱基基序。虽然UUA在整个三环构象中也显示出全局变化，但GUA除了缺少这个基序外，显示出与野生型非常相似的三环构象。缺少夹住碱基基序是这两个突变体与野生型之间唯一常见的结构差异。这些结果清楚地表明，UUA和GUA突变体的功能丧失主要源于一个小的关键识别基序的破坏，而不是源于它们三环构象的全局变化。基于这项研究，我们得出结论，复制酶识别的三环中的关键结构基序是一个在溶液中暴露的、夹住茎螺旋的三环中的5'-腺嘌呤碱基，这被称为夹住腺嘌呤基序。
