Kinetic analysis of the RNA-dependent adenosinetriphosphatase activity of DbpA, an Escherichia coli DEAD protein specific for 23S ribosomal RNA

被引:65
|
作者
Tsu, CA [1 ]
Uhlenbeck, OC [1 ]
机构
[1] Univ Colorado, Dept Chem & Biochem, Boulder, CO 80309 USA
关键词
D O I
10.1021/bi981837y
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The adenosinetriphosphatase (ATPase) activity of the Escherichia coli DEAD 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: k(cat) = 600 min(-1), K-app(RNA) 10 nM, and K-m(ATP) = 120 mu M. 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 10(3) to 10(6) [k(max)/K-app(RNA)]. The interaction with this small RNA fragment was further investigated with regard to stoichiometry, pH, salt and temperature. DbpA is not activated by E. coli ribosomes, 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.
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收藏
页码:16989 / 16996
页数:8
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