Structural mimicry in the phage [phis]21 N peptide–boxB RNA complex

Abstract

We determined the solution structure of a 22-amino-acid peptide from the amino-terminal domain of the bacteriophage 21 N protein in complex with its cognate 24-mer boxB RNA hairpin using heteronuclear magnetic resonance spectroscopy. The N peptide binds as an ␣-helix and interacts predominately with the major groove side of the 5 half of the boxB RNA stem-loop. This binding interface is defined by surface complementarity of polar and nonpolar interactions, and little sequence-specific recognition. The 21 boxB loop (CUAACC) has hydrogen bond and backbone torsions typical of the "U-turn" motif, as well as base stacking of the last 4 nt, and a hydrogen bonded C:C pair closing the loop. The exposed face of the 21 boxB loop, in complex with the N peptide, is strikingly similar to the GNRA tetraloop-like folds of the related and P22 bacteriophage N peptide-boxB RNA complexes. The N peptide-boxB complexes of the various phage, while individually distinct, provide similar structural features for interactions with the Escherichia coli host factors to enable antitermination.

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

“…Other work in vivo shows that boxB right has significant activity with P22 N in vivo (13,19) and that P22 boxB right is highly specific (13,28). Despite the uniform context of the boxBpeptide interaction, the specificities displayed by our boxB reporters largely agree with published data, though there are unresolved discrepancies between published boxB-N peptide specificities in vitro (2,7,43,44).…”

“…The boxBs display specificity for their cognate N proteins; the P22 boxB right reporter displays very high specificity. In vitro experiments indicate substantial affinity between boxB left and the P22 N peptide (44) and between P22 boxB left and the N peptide (7). Other work in vivo shows that boxB right has significant activity with P22 N in vivo (13,19) and that P22 boxB right is highly specific (13,28).…”

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.…”

“…For example single 15 N [Gly] labels were introduced into a 14-residue peptide to facilitate the assignments of three glycine residues [31]. However, bacterial expression is also frequently used to generate uniform 15 N or 15 N/ 13 C labeled peptides [6,7,9,19,33,42,44,165].…”

“…Through-bond connectivities between the anomeric protons H1ʹ′ and H8/H6 can be established with HCNCH experiments or indirectly via the 15 N chemical shift using HCN experiments [189,190] as was demonstrated on a 10 kDa peptide-RNA complex [9]. Recent developments using TROSY and multiplequantum (MQ)-transfers improve sensitivity of triple resonance experiments for RNA pushing the size limit to higher molecular weight [191].…”