RNA RECOGNITION BY A BENT ALPHA-HELIX REGULATES TRANSCRIPTIONAL ANTITERMINATION IN PHAGE-LAMBDA

A novel RNA recognition motif is characterized in an arginine-rich pep tide. The motif, derived from lambda transcriptional antitermination p rotein N, regulates an RNA-directed genetic switch. Its characterizati on by multidimensional nuclear magnetic resonance (NMR) demonstrates s pecific RNA-dependent folding of N- and C-terminal recognition helices separated by a central bend. The biological importance of the bent al pha-helix is demonstrated by mutagenesis: binding is blocked by substi tutions in the N peptide or its target (the boxB RNA hairpin) associat ed in vivo with loss of transcriptional antitermination activity. Alth ough arginine side chains are essential, the peptide is also anchored to boxB by specific nonpolar contacts. An alanine in the N-terminal he lix docks in the major groove of the RNA stem whereas a tryptophan in the C-terminal helix stacks against a purine in the RNA loop. At these positions all 19 possible amino acid substitutions have been construc ted by peptide synthesis; each impairs binding to boxB. The pattern of allowed and disallowed substitutions is in accord with the results of random-cassette mutagenesis in vivo. The helix-bend-helix motif ratio nalizes genetic analysis of N-dependent transcriptional antiterminatio n and extends the structural repertoire of arginine-rich domains obser ved among mammalian immunodeficiency viruses.