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Differential Modes of Recognition in N Peptide−BoxB Complexes

Austin, Ryan J. and Xia, Tianbing and Ren, Jinsong and Takahashi, Terry T. and Roberts, Richard W. (2003) Differential Modes of Recognition in N Peptide−BoxB Complexes. Biochemistry, 42 (50). pp. 14957-14967. ISSN 0006-2960. doi:10.1021/bi0351312.

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N proteins from bacteriophages λ, P22, and φ21 modulate transcription elongation by binding nascent “boxB” mRNA hairpins. This RNA recognition is mediated by N-terminal arginine-rich peptide sequences capable of interacting with their cognate boxB RNA targets. Here, we have analyzed the affinity and specificity of the peptide−RNA interactions that modulate this transcriptional switch. To do this, we constructed a series of peptides based on the wild-type λ, P22, and φ21 N protein binding domains ranging from 11 to 22 residues and analyzed their interactions with the leftward and rightward boxB RNA hairpin targets for all three phage. Binding constant (Kd) values were determined using RNA hairpins labeled with 2-aminopurine (2AP) and monitoring the fluorescence change as peptide was added. Kd's demonstrate that λ and P22 N peptides bind to their cognate boxB targets with high specificity and show equal affinities for their leftward and rightward hairpins. Surprisingly, φ21 shows very little specificity for its cognate targets. λ and P22 N peptides exhibit differential modes of recognition with specificity conferred by their amino- and carboxy-terminal modules, respectively. We have generated a reciprocal matrix of substituted peptides to examine the contributions of individual residues to specificity. Amino acid coupling analysis supports a binding model where the Arg8 residue of λ peptide acts as a conformational hot spot, anchoring the induced loop fold of its boxB hairpin target.

Item Type:Article
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URLURL TypeDescription ItemSupporting Information
Austin, Ryan J.0000-0002-5302-5266
Roberts, Richard W.0000-0002-8587-5097
Additional Information:© 2003 American Chemical Society. Received 1 July 2003. Published online 27 November 2003. Published in print 1 December 2003. This work was supported by funding from NSF Grant 9876246 (R.W.R.), NIH Grant RO160416 (R.W.R.), and Training Grant T32 GM08501. R.W.R. is an Alfred P. Sloan Foundation research fellow. We thank Jeffrey Barrick for suggestions and Dr. Adam Frankel (Caltech) for comments on the manuscript.
Funding AgencyGrant Number
NIH Predoctoral FellowshipT32 GM08501
Alfred P. Sloan FoundationUNSPECIFIED
Issue or Number:50
Record Number:CaltechAUTHORS:20170131-140940448
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Official Citation:Differential Modes of Recognition in N Peptide−BoxB Complexes Ryan J. Austin, Tianbing Xia, Jinsong Ren, Terry T. Takahashi, and Richard W. Roberts Biochemistry 2003 42 (50), 14957-14967 DOI: 10.1021/bi0351312
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:73888
Deposited By: Ruth Sustaita
Deposited On:01 Feb 2017 20:48
Last Modified:11 Nov 2021 05:23

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