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 singlestranded 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.Macromolecular machines involving multiple protein and nucleic acid components drive and regulate many important cellular processes. These include gene expression, protein synthesis and trafficking, as well as signal transduction and many other processes. The assembly of the relevant macromolecular complexes is often controlled by additional regulatory factors that modulate the effective binding affinity of the central components of a self-assembling macromolecular machine. Another regulatory strategy involves controlling the local concentration of a critical component of the complex, which can perturb crucial binding interactions by manipulating mass action effects. In principle this strategy uses changes in the concentration parameter as a "switch" to turn the targeted assembly reaction on or off; a good example of this strategy involves using the control of the synthesis (and thus of the free concentration) of the single-stranded DNA-binding protein of T4 (gp32) to regulate the assembly of the functional T4 DNA replication complex (1). Furthermore, if this component is "tethered" in the vicinity of the assembly to be regulated (e.g. by cis-RNA looping (2, 3)), this switch can be used to confine the regulation of concentration change only to nearby target loci on the genome and not to others.Characterizing the binding interactions between particular components of a macromolecular complex and elucidating the mechanisms of assembly is a challenging, but vital, part of understanding how cellular processes are regulated. This problem is compounded when...