SecA protein of Escherichia coli (E. coli), an ATPase essential for the translocation of precursor proteins, was found to have an additional activity of RNA helicase. This RNA unwinding activity of SecA was tested with two kinds of RNA duplex which have different predicted stability and nucleotide sequence. Each of these duplexes is consisted of two strands of unequal length with single-stranded ends. The RNA helicase activity of SecA required ATP and divalent cations. A monovalent cation such as K+ was ineffective for this activity. The helicase activities of NS3 protein of hepatitis C virus(HCV) and its deletion mutant were compared with that of SecA under the same condition. A clear confirmation of this SecA RNA helicase activity came from the inhibition of unwinding of the RNA duplex when SecA was preincubated with its own polyclonal antibody. The preimmune IgG could not inhibit the RNA unwinding by SecA.
Next, the RNA binding and the ATPase activity of SecA were studied. For this study, several different single and double strand RNAs were used. It was observed that the SecA protein binds to RNA regardless of its duplexity and nucleotide sequence. This nonspecificity of SecA protein for the RNA substrate was also observed in a previous result that SecA could unwind two kinds of RNA duplex. The ATPase activity of SecA was stimulated by the binding of RNA to the SecA protein. The effect of divalent cation concentration on the ATPase activity was the same as that on the RNA helicase activity of SecA.
Finally, the effect of precursor ribose binding protein (pRBP) on the RNA binding and the ATPase activity of SecA were studied. First, it was observed that the RNA binding efficiency of free SecA protein was greater than that of pRBP-bound SecA protein. Second, the extent of ATP hydrolysis by SecA increases when preincubated with RNA, but decreases when the refolding pRBP was added to the RNA-bound SecA protein.
Possible biological function of the SecA RNA heli...