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 Escherichia coli.
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.
Greive
Sandra J
SJ
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403, USA.
Lins
August F
AF
von Hippel
Peter H
PH
D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry; D001435:Bacteriophages / Q000378:metabolism; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D013058:Mass Spectrometry; D008954:Models, Biological; D008956:Models, Chemical; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D012335:RNA, Ribosomal / Q000737:chemistry*; D012269:Ribosomal Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry*; D014158:Transcription, Genetic; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C074952:NusB protein, E coli; D011506:Proteins; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D014157:Transcription Factors; C029537:ribosomal protein S10; D012313:RNA; D012321:DNA-Directed RNA Polymerases