A protein-RNA interaction network facilitates the template-independent cooperative assembly on RNA polymerase of a stable antitermination complex containing the lambda N protein.
The stable association of the N gene transcriptional antiterminator protein of bacteriophage lambda with transcribing RNA polymerase requires a nut site (boxA+boxB) in the nascent transcript and the Escherichia coli factors NusA, NusB, NusG, and ribosomal protein S10. We have used electrophoretic mobility shift assays to analyze the assembly of N protein, the E. coli factors, and RNA polymerase onto the nut site RNA in the absence of a DNA template. We show that N binds boxB RNA and that subsequent association of NusA with the N-nut site complex is facilitated by both boxA and boxB. In the presence of N, NusA, and RNA polymerase the nut site assembles ribonucleoprotein complexes containing NusB, NusG, and S10. The effects on assembly of mutations in boxA, boxB, NusA, and RNA polymerase define multiple weak protein-protein and protein-RNA interactions (e.g., NusB with NusG; NusA with boxB; NusA, NusB, and NusG with boxA) that contribute to the overall stability of the complex. Interaction of each component of the complex with two or more other components can explain the many observed cooperative binding associations in the DNA-independent assembly of a stable antitermination complex on RNA polymerase.
Mogridge
J
J
Banting and Best Department of Medical Research, University of Toronto, Canada.
Mah
T F
TF
Greenblatt
J
J
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D029968:Escherichia coli Proteins; D016153:Genes, Suppressor; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D035602:Transcriptional Elongation Factors; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C059417:N protein, Bacteriophage lambda; C074952:NusB protein, E coli; C070291:NusG protein, E coli; D009841:Oligonucleotides; D010445:Peptide Elongation Factors; D011506:Proteins; D012269:Ribosomal Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D054334:Viral Regulatory and Accessory Proteins; C068727:nusA protein, E coli; C029537:ribosomal protein S10; D012313:RNA; D012321:DNA-Directed RNA Polymerases