Antitermination in bacteriophage λ

Abstract

The solution structure of a 15-mer nutRboxB RNA hairpin complexed with the 36-mer N-terminal peptide of the N protein (N36) from bacteriophage lambda was determined by 2D and 3D homonuclear and heteronuclear magnetic resonance spectroscopy. These 36 amino acids include the arginine-rich motif of the N protein involved in transcriptional antitermination of phage lambda. Upon complex formation with boxB RNA, the synthetic N36 peptide binds tightly to the major groove of the boxB hairpin through hydrophobic and electrostatic interactions forming a bent alpha helix. Four nucleotides of the GAAAA pentaloop of the boxB RNA adopt a GNRA-like tetraloop fold in the complex. The formation of a GAAA tetraloop involves a loop-closing sheared base pair (G6-A10), base stacking of three adenines (A7, A8, and A10), and extrusion of one nucleotide (A9) from the loop, as observed previously for the complex of N(1-22) peptide and the nutLboxB RNA [Legault, P., Li, J., Mogridge, J., Kay, L.E. & Greenblatt, J. (1998) Cell 93, 289-299]. Stacking of the bases is extended by the indole-ring of Trp18 which also forms hydrophobic contacts to the side-chains of Leu24, Leu25, and Val26. Based on the structure of the complex, three mutant peptides were synthesized and investigated by CD and NMR spectroscopy in order to determine the role of particular residues for complex formation. These studies revealed very distinct amino-acid requirements at positions 3, 4, and 8, while replacement of Trp18 with tyrosine did not result in any gross structural changes.

The RNA–protein complex: Direct probing of the interfacial recognition dynamics and its correlation with biological functions

“…5, we show the biological in vivo activity and the amplitude of the ultrafast component for WT, KH, and ER. The stacking of (Upper) Stacked structure is a schematic representation of the WT complex from NMR studies (10,11). Domain motion of the C-terminal portion of N peptides switches the stacked structure to the unstacked structure.…”

“…NMR structural studies (10)(11)(12) demonstrated that the amino terminal of the N protein (N peptide), which features an arginine-rich motif, binds to the boxB RNA hairpin as a bent ␣-helix, and this process enforces the purine-rich boxB RNA pentaloop to adopt a canonical GNRA fold (13,14) with the fourth purine residue extruded (Fig. 1).…”

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].…”

“…Some structures of protein-RNA complexes are very precise (heavy atom RMSD below 1Å) because of use of a large number of NOEs, especially intermolecular NOEs. For example, the structure of the N peptide of the bacteriophage λ in complex with its BoxB RNA target is among the most precise NMR structure of a protein-RNA complex determined to date [33]. This complex has a molecular weight of 8.9 kDa and consists of a 36-residue peptide bound to a 15-nucleotide RNA.…”