Kinetic analysis of the RNA-dependent adenosinetriphosphatase activity of DbpA, an Escherichia coli DEAD protein specific for 23S ribosomal RNA.
Kinetic analysis of the RNA-dependent adenosinetriphosphatase activity of DbpA, an Escherichia coli DEAD protein specific for 23S ribosomal RNA.
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DbpA（一种对 23S 核糖体 RNA 具有特异性的大肠杆菌 DEAD 蛋白）的 RNA 依赖性三磷酸腺苷酶活性的动力学分析。
DOI：
10.1021/bi981837y
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Uhlenbeck,OC
中科院分区：
文献类型：
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作者：
Tsu,CA;Uhlenbeck,OC
大肠杆菌DEAD蛋白DbpA的腺苷三磷酸酶（ATPase）活性是不寻常的，因为它是由23 S核糖体RNA（rRNA）特异性刺激的。用ATP酶偶联光谱法研究了DbpA与RNA和ATP的相互作用。23 S rRNA结构域V的153个碱基片段在激活DbpA方面与完整的天然rRNA动力学相同：kcat= 600 min-1，Kapp（RNA）= 10 nM，Km（ATP）= 120 μM。在不存在RNA的情况下，ATP酶周转率为0.25 min-1。缺少这个位点的23 S rRNA片段（核苷酸2454 - 2606）基本上是无活性的，其他RNA如poly（A）和tRNA也是如此。DbpA的相对RNA特异性范围为103 ~ 106[kmax/Kapp（RNA）]。进一步研究了与该小RNA片段的相互作用与化学计量、pH、盐和温度的关系。DbpA不被大肠杆菌核糖体激活，也不被大亚基激活，而变性核糖体则刺激完整的ATP酶活性。结合紧密，位点特异性结合裸，未修饰的23 S rRNA，这表明DbpA在核糖体生物合成中的作用，而不是翻译。
The adenosinetriphosphatase (ATPase) activity of theEscherichia coliDEAD protein DbpA is unusual in that it is specifically stimulated by 23S ribosomal RNA (rRNA). A coupled spectroscopic ATPase assay was used to investigate the interaction of DbpA with RNA and ATP. A 153-base fragment of domain V of 23S rRNA is kinetically identical to intact, native rRNA in activating DbpA:kcat= 600 min-1,Kapp(RNA) = 10 nM, andKm(ATP) = 120 μM. The ATPase turnover in the absence of RNA is 0.25 min-1. Fragments of 23S rRNA lacking this site (nucleotides 2454−2606) are essentially inactive, as are other RNAs such as poly(A) and tRNA. The relative RNA specificity of DbpA ranges from 103to 106[kmax/Kapp(RNA)]. The interaction with this small RNA fragment was further investigated with regard to stoichiometry, pH, salt and temperature. DbpA is not activated byE.coliribosomes, nor by large subunits, while denatured ribosomes stimulate full ATPase activity. Taken together with the tight, site-specific binding to naked, unmodified 23S rRNA, this suggests a role for DbpA in ribosome biogenesis rather than translation.