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J. Biol. Chem., Vol. 280, Issue 37, 32177-32183, September 16, 2005

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Low Energy CD of RNA Hairpin Unveils a Loop Conformation Required for N Antitermination Activity^*

Neil P. Johnson1, Walter A. Baase, and Peter H. von Hippel2

From the Institut de Pharmacologie et de Biologie Structurale, UMR 5089, CNRS, 205 Route de Narbonne, 31077 Toulouse, France and the Department of Chemistry and Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229

N protein coded by phage is a transcription factor that stimulates the antitermination activity of Escherichia coli RNA polymerase by binding specifically to the nascent RNA transcript at a stemloop structure called boxB. We use a new biophysical technique, involving the monitoring of the low energy circular dichroism spectra of 2-aminopurine residues site-specifically placed in the boxB RNA loop, to investigate this binding interaction. The low energy CD spectra of these 2-aminopurine probes reflect specific asymmetric interactions with adjacent nucleotide bases. Consequently, these CD spectra provide detailed and specific conformational information about the RNA chain at these chromophores that cannot be obtained from changes in the related fluorescence signals of these probes. CD changes were observed on binding the N peptide to boxB RNA that correspond to structural changes that had been previously seen by NMR, thus validating our experimental approach. The low energy CD method was then used to quantify the ordered and disordered states of the free hairpin loop and to show that a significant fraction of the boxB loop assumes a product-like structure in the absence of protein. A boxB derivative with an intact stem and a reduced concentration of ordered loop was identified and used to show that the extent of the reaction between protein and boxB depends on the concentration of structured loop in the RNA reactant population. This result has general implications for the conformational specificity of RNA-protein interactions.

Received for publication, April 27, 2005 , and in revised form, July 14, 2005.

^* This work was supported in part by National Institutes of Health Grants GM-15792 and GM-29158 (to P. H. v. H.) and by partial sabbatical salary support from CNRS (to N. P. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed. E-mail: Neil.Johnson{at}ipbs.fr. ² An American Cancer Society Research Professor of Chemistry. To whom correspondence may be addressed. E-mail: petevh{at}molbio.uoregon.edu.

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