Analysis of bacteriophage N protein and peptide binding to boxB RNA using polyacrylamide gel coelectrophoresis (PACE).
The antitermination protein N from bacteriophage lambda (Nlambda) 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 Nlambda protein is known to interact with a small RNA hairpin (boxB) derived from the nut site RNA. We have examined the binding of Nlambda protein, peptides derived from the amino terminus of Nlambda, and the related phage P22 N protein to lambda 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 Nlambda protein binds lambda boxB RNA with a Kd,app of 5.2 nM. PACE was also used to study the binding affinity of a number of Nlambda peptide and lambda boxB 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.
Cilley
C D
CD
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Williamson
J R
JR
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D017830:Octoxynol; D010446:Peptide Fragments / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D012367:RNA, Viral / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012965:Sodium Chloride / Q000494:pharmacology; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry / Q000378:metabolism*
D003902:Detergents; D046911:Macromolecular Substances; C059417:N protein, Bacteriophage lambda; D010446:Peptide Fragments; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D054334:Viral Regulatory and Accessory Proteins; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D017830:Octoxynol; C010615:Nonidet P-40