The antitermination protein N from bacteriophage λ (Nλ)
interacts with the nut site in its own mRNA, as
well as host factors, to facilitate formation of a termination-resistant
transcription complex. The conserved, amino-terminal arginine-rich
domain of Nλ protein is known to interact
with a small RNA hairpin (boxB) derived from the
nut site RNA. We have examined the binding of
Nλ protein, peptides derived from the amino
terminus of Nλ, and the related phage P22
N protein to λ boxB RNAs. To facilitate the
study of complexes that are not amenable to gel retardation
assays, a new polyacrylamide affinity coelectrophoresis
technique (PACE) was developed. Using the PACE assay, we
have demonstrated that a 19-amino acid peptide from the
amino terminus of Nλ protein binds λ
boxB RNA with a Kd,app
of 5.2 nM. PACE was also used to study the binding affinity
of a number of Nλ peptide and λboxB
RNA mutants. The PACE technique is complementary to the
traditional gel retardation assay for direct measurement
of binding interactions, and will be useful for any procedure
that requires a pool of RNAs to be resolved based on their
relative affinities for proteins or peptides.