Rapid steps in the glmS ribozyme catalytic pathway: cation and ligand requirements.
Rapid steps in the glmS ribozyme catalytic pathway: cation and ligand requirements.
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GLMS核酶催化途径的快速步骤：阳离子和配体要求。
DOI：
10.1021/bi101842u
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发表时间：
2011-04-05
期刊：
影响因子：
2.9
通讯作者：
Hampel KJ
中科院分区：
文献类型：
--
作者：
Brooks KM;Hampel KJ
GLMS核酶是一种保守的核糖开关，存在于许多革兰氏阳性细菌中，对细胞内氨基葡萄糖-6-磷酸(GlcN6P)的浓度做出反应。GlcN6P结合促进GLMS基因5‘端非编码区的特异性自我切割，导致基因表达下调。以前已经证明，当测量切割反应途径的限速折叠步骤时，GLMS核酶缺乏强阳离子特异性。这没有提供关于GLMS核酶在快速配体结合化学催化事件中的阳离子和配体特异性的数据。核酶在含镁缓冲液中的预折叠有效地将快速配体结合和催化事件(Kobs>；60min−1)从限速折叠(Kobs<；4min−1)中分离出来。在这里，我们采用这个实验设计来分析快速配体结合和催化所需的阳离子和配体。我们发现，一价阳离子的摩尔浓度也能诱导天然的GlcN6P结合结构的形成，但不能促进配体结合和催化速率>；4min−1。我们的数据表明，有结晶学证据的二价阳离子的唯一必需作用是配位配体结合口袋中的GlcN6P的磷酸部分。为了进一步支持这一假说，我们的数据表明，GlcN6P的非磷酸化类似物氨基葡萄糖在二价阳离子存在的情况下不能促进快速的配体结合和催化。因此，核酶的折叠相对独立于阳离子的同一性，但添加配体后催化的快速启动更为严格。
The glmS ribozyme is a conserved riboswitch found in numerous Gram-positive bacteria and responds to the cellular concentrations of glucosamine-6-phosphate (GlcN6P). GlcN6P binding promotes site-specific self-cleavage in the 5′UTR of the glmS mRNA, resulting in down regulation of gene expression. The glmS ribozyme has previously been shown to lack strong cation specificity when the rate-limiting folding step of the cleavage reaction pathway is measured. This does not provide data regarding cation and ligand specificities of the glmS ribozyme during the rapid ligand binding chemical catalysis events. Prefolding of the ribozyme in Mg2+-containing buffers effectively isolates the rapid ligand binding and catalytic events (kobs > 60 min−1) from rate-limiting folding (kobs<4 min−1). Here we employ this experimental design to assay the cations and ligand requirements for rapid ligand binding and catalysis. We show that molar concentrations of monovalent cations are also capable of inducing the formation of the native GlcN6P binding structure, but are unable to promote ligand binding and catalysis rates >4 min−1. Our data show that the sole obligatory role for divalent cations, for which there is crystallographic evidence, is coordination of the phosphate moiety of GlcN6P in the ligand binding pocket. In further support of this hypothesis, our data show that a non-phosphorylated analog of GlcN6P, glucosamine, is unable to promote rapid ligand binding and catalysis in the presence of divalent cations. Folding of the ribozyme is, therefore, relatively independent of cation identity but the rapid initiation of catalysis upon the addition of ligand is stricter.
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影响因子：
3.2
作者：
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通讯作者：
Soukup, GA