Ca. Tsu et Oc. Uhlenbeck, Kinetic analysis of the RNA-dependent adenosinetriphosphatase activity of DbpA, an Escherichia coli DEAD protein specific for 23S ribosomal RNA, BIOCHEM, 37(48), 1998, pp. 16989-16996
The adenosinetriphosphatase (ATPase) activity of the Escherichia coli DEAD
protein DbpA is unusual in that it is specifically stimulated by 23S riboso
mal RNA (rRNA). A coupled spectroscopic ATPase assay was used to investigat
e 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 rRN
A lacking this site (nucleotides 2454-2606) are essentially inactive, as ar
e 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, p
H, 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 2
3S rRNA, this suggests a role for DbpA in ribosome biogenesis rather than t
ranslation.