Hydrodynamic characterization of the DEAD-box RNA helicase DbpA

被引:18
作者
Talavera, MA
Matthews, EE
Eliason, WK
Sagi, I
Wang, J
Henn, A
De La Cruz, EM [1 ]
机构
[1] Yale Univ, Dept Mol Biophys & Biochem, New Haven, CT 06520 USA
[2] Yale Univ, Dept Chem, New Haven, CT 06520 USA
[3] Weizmann Inst Sci, Dept Biol Struct, IL-76100 Rehovot, Israel
关键词
DbpA; helicase; RNA; analytical ultracontrifugation; bead modeling;
D O I
10.1016/j.jmb.2005.10.058
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-cross-exclusion chromatography, analytical ultracentrifugation, chemical linking and hydrodynamic modeling. DbpA is monomeric in solution up to a concentration of 25 mu M and over the temperature range of 4 degrees C to 22 degrees C. Binding of neither nucleoticle (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 23 S rRNA and interacts specifically with hairpin 92 of the PTC. (c) 2005 Elsevier Ltd. All rights reserved.
引用
收藏
页码:697 / 707
页数:11
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