Kinetic analysis of the RNA-dependent adenosinetriphosphatase activity of DbpA, an Escherichia coli DEAD protein specific for 23S ribosomal RNA.
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: kcat = 600 min-1, Kapp(RNA) = 10 nM, and Km(ATP) = 120 microM. 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) [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 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.
Tsu
C A
CA
Department of Chemistry and Biochemistry, Campus Box 215, University of Colorado, Boulder, Colorado 80309-0215, USA.
Uhlenbeck
O C
OC
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D053487:DEAD-box RNA Helicases; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D020365:RNA Helicases / Q000378:metabolism*; D012329:RNA, Bacterial / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000378:metabolism*; D016601:RNA-Binding Proteins; D012270:Ribosomes / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D016601:RNA-Binding Proteins; D000251:Adenosine Triphosphatases; C065908:dbpA protein, E coli; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases