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Journal of Bacteriology, December 2008, p. 7699-7708, Vol. 190, No. 23
0021-9193/08/$08.00+0     doi:10.1128/JB.00997-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The RNA-Binding Domain of Bacteriophage P22 N Protein Is Highly Mutable, and a Single Mutation Relaxes Specificity toward {lambda}{triangledown}

Alexis I. Cocozaki, Ingrid R. Ghattas, and Colin A. Smith*

Department of Biology, American University of Beirut, Beirut, Lebanon

Received 19 July 2008/ Accepted 12 September 2008

Antitermination in bacteriophage P22, a lambdoid phage, uses the arginine-rich domain of the N protein to recognize boxB RNAs in the nut site of two regulated transcripts. Using an antitermination reporter system, we screened libraries in which each nonconserved residue in the RNA-binding domain of P22 N was randomized. Mutants were assayed for the ability to complement N-deficient virus and for antitermination with P22 boxBleft and boxBright reporters. Single amino acid substitutions complementing P22 N– virus were found at 12 of the 13 positions examined. We found evidence for defined structural roles for seven nonconserved residues, which was generally compatible with the nuclear magnetic resonance model. Interestingly, a histidine can be replaced by any other aromatic residue, although no planar partner is obvious. Few single substitutions showed bias between boxBleft and boxBright, suggesting that the two RNAs impose similar constraints on genetic drift. A separate library comprising only hybrids of the RNA-binding domains of P22, {lambda}, and {phi}21 N proteins produced mutants that displayed bias. P22 N– plaque size plotted against boxBleft and boxBright reporter activities suggests that lytic viral fitness depends on balanced antitermination. A few N proteins were able to complement both {lambda} N- and P22 N-deficient viruses, but no proteins were found to complement both P22 N- and {phi}21 N-deficient viruses. A single tryptophan substitution allowed P22 N to complement both P22 and {lambda} N–. The existence of relaxed-specificity mutants suggests that conformational plasticity provides evolutionary transitions between distinct modes of RNA-protein recognition.

* Corresponding author. Mailing address: Department of Biology, American University of Beirut, P.O. Box 11-0236, Riad El Solh 1107 2020, Beirut, Lebanon. Phone: 961 3 791313, ext. 3887. Fax: 961 1 744 461. E-mail: cs10{at}aub.edu.lb

{triangledown} Published ahead of print on 26 September 2008.

Journal of Bacteriology, December 2008, p. 7699-7708, Vol. 190, No. 23
0021-9193/08/$08.00+0     doi:10.1128/JB.00997-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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