Mutational Analysis of the Escherichia coli DEAD Box Protein CsdA

Abstract

ABSTRACT The Escherichia coli cold shock protein CsdA is a member of the DEAD box family of ATP-dependent RNA helicases, which share a core of nine conserved motifs. The DEAD (Asp-Glu-Ala-Asp) motif for which this family is named has been demonstrated to be essential for ATP hydrolysis. We show here that CsdA exhibits in vitro ATPase and helicase activities in the presence of short RNA duplexes with either 3′ or 5′ extensions at 15°C. In contrast to wild-type CsdA, a DQAD variant of CsdA (Glu-157→Gln) had no detectible helicase or ATPase activity at 15°C in vitro. A plasmid encoding the DQAD variant was also unable to suppress the impaired growth of the csdA null mutant at 15°C. Plasmid-encoded CsdAΔ444, which lacks most of the carboxy-terminal extension, enhanced the growth of a csdA null mutant at 25°C but not at 15°C; this truncated protein also has limited in vitro activity at 15°C. These results support the physiological function of CsdA as a DEAD box ATP-dependent RNA helicase at low temperature.