The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA

J Mol Biol. 2008 Mar 14;377(1):193-205. doi: 10.1016/j.jmb.2007.12.046. Epub 2007 Dec 28.

Abstract

DEAD-box proteins are ATPase enzymes that destabilize and unwind duplex RNA. Quantitative knowledge of the ATPase cycle parameters is critical for developing models of helicase activity. However, limited information regarding the rate and equilibrium constants defining the ATPase cycle of RNA helicases is available, including the distribution of populated biochemical intermediates, the catalytic step(s) that limits the enzymatic reaction cycle, and how ATP utilization and RNA interactions are linked. We present a quantitative kinetic and equilibrium characterization of the ribosomal RNA (rRNA)-activated ATPase cycle mechanism of DbpA, a DEAD-box rRNA helicase implicated in ribosome biogenesis. rRNA activates the ATPase activity of DbpA by promoting a conformational change after ATP binding that is associated with hydrolysis. Chemical cleavage of bound ATP is reversible and occurs via a gamma-phosphate attack mechanism. ADP-P(i) and RNA binding display strong thermodynamic coupling, which causes DbpA-ADP-P(i) to bind rRNA with >10-fold higher affinity than with bound ATP, ADP or in the absence of nucleotide. The rRNA-activated steady-state ATPase cycle of DbpA is limited both by ATP hydrolysis and by P(i) release, which occur with comparable rates. Consequently, the predominantly populated biochemical states during steady-state cycling are the ATP- and ADP-P(i)-bound intermediates. Thermodynamic linkage analysis of the ATPase cycle transitions favors a model in which rRNA duplex destabilization is linked to strong rRNA and nucleotide binding. The presented analysis of the DbpA ATPase cycle reaction mechanism provides a rigorous kinetic and thermodynamic foundation for developing testable hypotheses regarding the functions and molecular mechanisms of DEAD-box helicases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Triphosphatases / metabolism*
  • Adenosine Triphosphate / metabolism
  • Adenylyl Imidodiphosphate / analogs & derivatives
  • Adenylyl Imidodiphosphate / metabolism
  • DEAD-box RNA Helicases / metabolism*
  • Escherichia coli / enzymology*
  • Escherichia coli Proteins / metabolism*
  • Hydrolysis
  • Kinetics
  • Oxygen Isotopes
  • Peptidyl Transferases / metabolism
  • Phosphates / metabolism
  • RNA, Bacterial / metabolism
  • RNA, Ribosomal / metabolism
  • Thermodynamics

Substances

  • 2'(3')-O-methylanthraniloyl-gamma-amido-adenosine triphosphate
  • Escherichia coli Proteins
  • Oxygen Isotopes
  • Phosphates
  • RNA, Bacterial
  • RNA, Ribosomal
  • Adenylyl Imidodiphosphate
  • Adenosine Triphosphate
  • Peptidyl Transferases
  • Adenosine Triphosphatases
  • dbpA protein, E coli
  • DEAD-box RNA Helicases