title: Differential modes of recognition in N peptide-boxB complexes.
abstract: N proteins from bacteriophages lambda, P22, and phi21 modulate transcription elongation by binding nascent "boxB" mRNA hairpins. This RNA recognition is mediated by N-terminal arginine-rich peptide sequences capable of interacting with their cognate boxB RNA targets. Here, we have analyzed the affinity and specificity of the peptide-RNA interactions that modulate this transcriptional switch. To do this, we constructed a series of peptides based on the wild-type lambda, P22, and phi21 N protein binding domains ranging from 11 to 22 residues and analyzed their interactions with the leftward and rightward boxB RNA hairpin targets for all three phage. Binding constant (K(d)) values were determined using RNA hairpins labeled with 2-aminopurine (2AP) and monitoring the fluorescence change as peptide was added. K(d)'s demonstrate that lambda and P22 N peptides bind to their cognate boxB targets with high specificity and show equal affinities for their leftward and rightward hairpins. Surprisingly, phi21 shows very little specificity for its cognate targets. Lambda and P22 N peptides exhibit differential modes of recognition with specificity conferred by their amino- and carboxy-terminal modules, respectively. We have generated a reciprocal matrix of substituted peptides to examine the contributions of individual residues to specificity. Amino acid coupling analysis supports a binding model where the Arg8 residue of lambda peptide acts as a conformational hot spot, anchoring the induced loop fold of its boxB hairpin target.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Austin
authors.forename: Tianbing
authors.initials: T
authors.lastname: Xia
authors.forename: Jinsong
authors.initials: J
authors.lastname: Ren
authors.forename: Terry T
authors.initials: TT
authors.lastname: Takahashi
authors.forename: Richard W
authors.initials: RW
authors.lastname: Roberts
mesh_terms: D000595:Amino Acid Sequence; D017100:Bacteriophage P22 / Q000737:chemistry / Q000378:metabolism; D010582:Bacteriophage lambda / Q000737:chemistry / Q000378:metabolism; D002151:Calorimetry; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D011485:Protein Binding; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013816:Thermodynamics; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: C059417:N protein, Bacteriophage lambda; D019590:Nucleocapsid Proteins; D010446:Peptide Fragments; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D054334:Viral Regulatory and Accessory Proteins