Fig 8.

Differential binding of wild-type and mutant aconitase proteins to the citZ 5′ leader RNA in vitro. Each of six Acn protein preparations (described in the legend of Fig. 2) were mixed separately at the indicated concentrations with radiolabeled citZ 5′ leader RNA synthesized in vitro (83 pM). Reactions were allowed to equilibrate in buffer containing RNase inhibitor (RNaseOut; Invitrogen), dipyridyl (0.5 mM), and β-mercaptoethanol (5 mM) prior to passage through nitrocellulose membranes. RNA retained on each membrane was detected by scintillation counting, and the fraction of RNA retained was calculated, after background subtraction, as a percentage of the input RNA. The data shown are from a single experiment; wild-type preparation 2, C450S preparation 1, and R741E preparation 1 were assayed in other experiments and gave similar results.