Skip to main content
. 2006 Jan 5;34(1):10–22. doi: 10.1093/nar/gkj403

Figure 6.

Figure 6

RNA duplex formation by Ddx42p. (A) Ddx42p mediates annealing of complementary regions in RNA. RNA strands containing complementary regions of differing length were used as substrates for Ddx42p-catalyzed annealing reactions. The ss and ds control samples were incubated under identical conditions, but in the absence of Ddx42p. RNA annealing samples were subjected to SDS–PAGE and autoradiographed. D, M and P7 denote the partially ds product of the respective annealing reaction. The efficiency of RNA annealing by Ddx42p under helicase conditions increases with increasing length of the base pairing region: M (14 bp) < D (46 bp) < P7 (123 bp). The positions of the respective dsRNA and ssRNA species are indicated on the right. (B) Ddx42p-mediated RNA annealing is most efficient with ADP as the nucleotide cofactor. Annealing reactions with 50 nM Ddx42p were carried out with ssRNAs DT7 and DSP6 (complementary region of 46 bases) to exclude a possible counteracting of Ddx42p helicase activity. The reaction is most efficient with ADP, indicating that ATP must be hydrolyzed before RNA annealing. The weak RNA duplex band in lane 3 is attributed to 10% ADP content in the ATPγS preparation as declared by the manufacturer. (C) RNA annealing as a function of Ddx42p concentration. Formation of duplex D (46 bp) was monitored at increasing Ddx42p concentrations. Each point is the average of at least three measurements. In the presence of ADP (after ATP hydrolysis), significant annealing activity was observed even at a Ddx42p concentration used in helicase assays. (D) Ddx42p removes bound protein from RNA. Ddx42p-mediated RNA annealing reactions were carried out with the indicated nucleotide cofactors in the presence of RNA coated with saturating concentrations of the ss binding protein T4gp32. T4gp32 affects the Ddx42p-mediated RNA annealing reaction only when a 50-fold molar excess of T4gp32 over Ddx42p is used in the presence of ATP (lane 6). ADP-bound Ddx42p is even more effective as essentially no inhibition of annealing is seen under these conditions (lane 9).