Abstract
Analytical ultracentrifugation and fluorescence anisotropy methods have been used to measure the equilibrium parameters that control the formation of the core subcomplex of NusB and NusE proteins and boxA RNA. This subcomplex, in turn, nucleates the assembly of the antitermination complex that is involved in controlling the synthesis of ribosomal RNA in Escherichia coli and that also participates in forming the N protein-dependent antitermination complex in lambdoid phage synthesis. In this study we determined the dissociation constants (K(d) values) for the individual binary interactions that participate in the assembly of the ternary NusB-NusE-boxA RNA subassembly, and we showed that multiple equilibria, involving both specific and nonspecific binding, are involved in the assembly pathway of this protein-RNA complex. The measured K(d) values were used to model the in vitro assembly reaction and combined with in vivo concentration data to simulate the overall control of the assembly of this complex in E. coli at two different cellular growth rates. The results showed that at both growth rates assembly proceeds via the initial formation of a weak but specific NusB-boxA complex, which is then stabilized by NusE binding. We showed that NusE also binds nonspecifically to available single-stranded RNA sequences and that such nonspecific protein binding to RNA can help to regulate crucial interactions in the assembly of the various macromolecular machines of gene expression.
Highlights
When NusE was present in initial excess relative to boxA RNA as the concentration of NusB was increased, the measured equilibrium constants suggest that assembly of the NusB-NusE complex probably does occur first, followed by binding of the protein heterodimer to boxA RNA
This study showed that NusB binds and with relatively equal affinity, to both rrn RNA and boxA RNA (Fig. 6)
Having purified soluble NusE, and despite the potential complication introduced by scattered light, this study has shown that NusE binds to RNA nonspecifically and with a Kd of ϳ3 M and that this protein increases the affinity of NusB for boxA RNA by ϳ10-fold
Summary
Buffers and Reagents—All fluorescence anisotropy titrations and analytical ultracentrifugation analyses were conducted in buffer A (25 mM Hepes, 100 mM potassium acetate, pH 7.5), unless otherwise stated. The cell pellets were resuspended in buffer A (25 mM Hepes, pH 7.5, 100 mM potassium acetate) containing EDTA-free protease inhibitor mixture (Roche Applied Science) and 1 mM dithiothreitol (for NusE) and lysed by three passes through a French press at 1000 p.s.i. The resulting lysates were centrifuged at 10,000 ϫ g for 30 min to remove cell debris, and the supernatants were processed as described below. Fractions containing purified NusE from the second column were pooled and dialyzed against a 6 – 0 M urea gradient in buffer A over 24 h, after which precipitated NusE was removed by centrifugation and aliquots of the NusE protein snapfrozen and stored at Ϫ80 °C. Fractions containing NusB were identified by SDS-PAGE analysis, pooled, dialyzed against buffer A, and digested with TEV protease overnight at 16 °C, followed by dialysis against buffer A. University of California, Berkeley), and data from both analyses of the complex equilibria were plotted using the Profit program
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