Assembly of an RNA-protein complex: Binding of NusB and NusE (S10) proteins to boxA RNA nucleates the formation of the antitermination complex involved in controlling rRNA transcription in E. coli

doi:10.1074/jbc.M507146200

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Papers In Press, published online ahead of print August 18, 2005
J. Biol. Chem, 10.1074/jbc.M507146200
Submitted on June 30, 2005
Revised on August 17, 2005
Accepted on August 18, 2005

Assembly of an RNA-protein complex: Binding of NusB and NusE (S10) proteins to boxA RNA nucleates the formation of the antitermination complex involved in controlling rRNA transcription in E. coli

Sandra J. Greive, August F. Lins, and Peter H. von Hippel

Institute of Molecular Biology, University of Oregon, Eugene, OR 97403

Corresponding Author: petevh{at}molbio.uoregon.edu

Analytical ultracentrifugation and fluorescence anisotropy methods have been used to measure the equilibrium parameters that control the formation of the core sub-complex 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 E. coli and that also participates in forming the N protein-dependent antitermination complex in lambdoid phage synthesis. In this study we determine the dissociation constants (Kd values) for the individual binary interactions that participate in the assembly of the ternary NusB-NusE-boxA RNA sub-assembly, and show that multiple equilibria, involving both specific and nonspecific binding, are involved in the assembly pathway of this protein-RNA complex. The measured Kd values are 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 the E. coli at two different cellular growth rates. The results show that at both growth rates assembly proceeds via the initial formation of a weak but specific NusBboxA complex, which is then stabilized by NusE binding. We show 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.

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