FIGURE 1.
Stoichiometric and functional analysis of RraA complex with DEAD-box helicases SrmB and RhlB. The mass distribution for helicases, RraA, and the mixtures are shown in blue, red, and black, respectively. A–C, analytical ultracentrifugation of RhlB, SrmB, and SrmB(1–394) in the presence and absence of RraA. D, effects of RraA on the ATPase activity of SrmB. White and dark gray bars represent reactions with 24-mer RNA substrate in the absence and presence of RraA, respectively. Reactions with a mixture of 23S and 16S rRNA in the absence and presence of RraA are represented by light gray bars and bars filled with diagonal stripes. When present, RraA was added in 3-fold molar excess to helicase. Activity is expressed as mol of Pi/min/mol of protein. Each bar represents averaged values from at least three independent experiments, and error bars represent 2 S.D. E, interactions of SrmB and RraA evaluated by native polyacrylamide gel elecrophoresis. Free RraA migrates with the buffer front, but due to its positive charge, free SrmB does not enter the gel. The band corresponding to a complex of the two proteins (marked with asterisk) was extracted and analyzed by SDS-PAGE, confirming the presence of both RraA and SrmB.