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

Christopher A. Tsu, Olke C. Uhlenbeck*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

65 Scopus citations

Abstract

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 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 103 to 106 [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.

Original languageEnglish (US)
Pages (from-to)16989-16996
Number of pages8
JournalBiochemistry
Volume37
Issue number48
DOIs
StatePublished - Dec 1 1998

ASJC Scopus subject areas

  • Biochemistry

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