A LOOK INTO THE ATPase ACTIVE SITE OF THE DEAD BOX PROTEIN RNA HELIXCASE YIXN BY EPR SPECTROSCOPY

YxiN, is a DEAD box RNA helicase of Bacillus subtilis, which remodels RNA in an ATP dependent manner. It shows high specificity for Hairpin-92 ribosomal RNA (HP92). Its mechanism consists binding of RNA and Mg(II)ATP that results in ATP hydrolysis and RNA remodeling accompanied by large conformational changes in the protein [1]. While a general idea of this mechanism is known in terms of the kinetics involved, the structural basis for the coupling of the ATPase activity [2], the protein conformational changes and the RNA remodeling is unknown. In order to follow the steps of the RNA unwinding cycle we have studied the ATPase activity using EPR techniques where we replaced the Mg(II) with Mn(II). This substitution retains the helicase activity. We have focused on the ³¹P hyperfine coupling of the nucleotides with the Mn(II), which is a signature of the changes the Mn(II)ATP undergoes at the ATPase active site during the helicase function. This was determined by high field electron-nuclear double resonance (ENDOR) and electron-electron double resonance (ELDOR) detected NMR. We looked at various combinations of YixN/HP92/Mn (II) nucleotides (ADP, ATP and AMP-PNP). We found out that the behavior of YxiN is significantly different than that of DbpA, its analog from E.coli [3] and detected a unique state of post hydrolysis Mn(II)ADP binding site that occurs only when the RNA is bound to the protein.

[1] Kaminker, I.; Sushenko, A.; Potapov, A.; Daube, S.; Akabayov, B.; Sagi, I.; Goldfarb, D. J. Am. Chem. Soc. 2011, 133 (39), 15514-15523.

[2] Hilbert, M.; Karrow, A. R.; Klostermeier, D. Biol. Chem. 2009, 390, 1237-1250.

[3] Iost, I.; Dreyfus, M. Nucleic Acids Res. 2006, 34, 4189-4197.