Solution structure of P22 transcriptional antitermination N peptide–box B RNA complex

Abstract

We have determined the solution structure of a 15-mer boxB RNA hairpin complexed with a 20-mer basic peptide of the N protein involved in bacteriophage P22 transcriptional antitermination. Complex formation involves adaptive binding with the N peptide adopting a bent alpha-helical conformation that packs tightly through hydrophobic and electrostatic interactions against the major groove face of the boxB RNA hairpin, orienting the open opposite face for potential interactions with host factors and/or RNA polymerase. Four nucleotides in the boxB RNA hairpin pentaloop form a stable GNRA like tetraloop structural scaffold on complex formation, allowing the looped out fifth nucleotide to make extensive hydrophobic contacts with the bound peptide. The guanidinium group of a key arginine is hydrogen-bonded to the guanine in a loop-closing sheared G.A mismatch and to adjacent backbone phosphates. The identified intermolecular contacts account for the consequences of N peptide and boxB RNA mutations on bacteriophage transcriptional antitermination.

Bacteriophage P22 Antitermination boxB Sequence Requirements Are Complex and Overlap with Those of λ

“…NMR data support a model in which the N peptides recognize a specific conformation of boxB, with little direct recognition of the sequence (5,29,39). Most observed contacts are not sequence specific but are made to the backbone or are hydrophobic in nature.…”

“…1QFQ [39]), the NMR structure of P22 N peptide-P22 boxB left (Protein Data Bank accession no. 1A4T [5]), and the X-ray crystal structure of a Haloarcula marismortui large ribosomal subunit (Protein Data Bank accession no. 1JJ2 [27]).…”

“…No other proposed peptidebase hydrogen bond is supported by mutagenesis. Cai et al (5) report possible hydrogen bonding between a P22 N lysine and the base pair adjacent to the loop of P22 boxB left , Legault et al (29) propose hydrogen bonds between the N peptide and a base adjacent to the loop of boxB left , while Scharpf et al (39) have evidence only for hydrogen bonds to the guanine of the sheared G:A base pair of boxB right . -P22 specificity may reflect conformational energetics, likely governed by subtle base-stacking interactions.…”

“…Genetic and biochemical studies of the P22 interaction are less complete (13,44) but are supported by the solution state NMR structure of the arginine-rich domain of the N protein bound to P22 boxB right (5).…”

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

“…Alternatively, the affinity purification tag can be separated from the protein of interest. In the case of small peptides, removal of the affinity purification tag was often achieved using cyanogen bromide that specifically hydrolyzes peptide bonds C-terminal of methionines [7,9,15,16,19,33,42,44]. In the case of protein domains that often contain internal methionines, other methods for cleavage are generally used and involve specific proteases.…”