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 pr otein of bacteriophage lambda with transcribing RNA polymerase require s a nut site (boxA + boxB) in the nascent transcript and the Escherich ia coli factors NusA, NusB, NusG, and ribosomal protein S10. We have u sed electrophoretic mobility shift assays to analyze the assembly of N protein, the E. coli factors, and RNA polymerase onto the nut site RN A 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 fac ilitated by both boxA and boxB. In the presence of N, NusA, and RNA po lymerase the nut site assembles ribonucleoprotein complexes containing NusB, NusG, and S10. The effects on assembly of mutations in boxA, bo xB, 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 bindin g associations in the DNA-independent assembly of a stable antitermina tion complex on RNA polymerase.