Antitermination in bacteriophage lambda - The structure of the N36 peptide-boxB RNA complex

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 resonanc e spectroscopy. These 36 amino acids include the arginine-rich motif of the N protein involved in transcriptional antitermination of phage lambda. Upo n complex formation with boxB RNA, the synthetic N36 peptide binds tightly to the major groove of the boxB hairpin through hydrophobic and electrostat ic interactions forming a bent alpha helix. Four nucleotides of the GAAAA p entaloop of the boxB RNA adopt a GNRA-like tetraloop fold in the complex. T he 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 comple x 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 bas es is extended by the indole-ring of Trp18 which also forms hydrophobic con tacts to the side-chains of Leu24, Leu25, and Val26. Based on the structure of the complex, three mutant peptides were synthesiz ed and investigated by CD and NMR spectroscopy in order to determine the ro le of particular residues for complex formation. These studies revealed ver y distinct amino-acid requirements at positions 3, 4, and 8, while replacem ent of Trp18 with tyrosine did not result in any gross structural changes.