Hydrodynamic characterization of the DEAD-box RNA helicase DbpA.
The Escherichia coli DEAD-box protein A (DbpA) belongs to the highly conserved superfamily-II of nucleic acid helicases that play key roles in RNA metabolism. A central question regarding helicase activity is whether the process of coupling ATP hydrolysis to nucleic acid unwinding requires an oligomeric form of the enzyme. We have investigated the structural and functional properties of DbpA by multi-angle laser light-scattering, size-exclusion chromatography, analytical ultracentrifugation, chemical cross-linking and hydrodynamic modeling. DbpA is monomeric in solution up to a concentration of 25 microM and over the temperature range of 4 degrees C to 22 degrees C. Binding of neither nucleotide (ATP or ADP) nor peptidyl transferase center (PTC) RNA, the presumed physiological RNA substrate, favor oligomerization. The hydrodynamic parameters were used together with hydrodynamic bead modeling and structural homology in conjunction with ab initio structure prediction methods to define plausible shapes of DbpA. Collectively, the results favor models where DbpA functions as an active monomer that possesses two distinct RNA binding sites, one in the helicase core domain and the other in the carboxyl-terminal domain that recognizes 23S rRNA and interacts specifically with hairpin 92 of the PTC.
Talavera
Miguel A
MA
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06520, USA.
Matthews
Erin E
EE
Eliason
William K
WK
Sagi
Irit
I
Wang
Jimin
J
Henn
Arnon
A
De La Cruz
Enrique M
EM
D020816:Amino Acid Motifs; D002850:Chromatography, Gel; D003201:Computers; D003432:Cross-Linking Reagents / Q000737:chemistry; D053487:DEAD-box RNA Helicases; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D020365:RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D040681:Structural Homology, Protein
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D016601:RNA-Binding Proteins; D012313:RNA; C065908:dbpA protein, E coli; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases