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J Mol Biol. 2006 Sep 15;362(2):271-86. Epub 2006 Jul 22.

Flexibility and adaptability in binding of E. coli cytidine repressor to different operators suggests a role in differential gene regulation.

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Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA.


Interactions between DNA-bound transcription factors CytR and CRP regulate the promoters of the Escherichia coli CytR regulon. A distinctive feature of the palindromic CytR operators is highly variable length central spacers (0-9 bp). Previously we demonstrated distinct modes of CytR binding to operators that differ in spacer length. These different modes are characterized by opposite enthalpic and entropic contributions at 25 degrees C. Of particular note were radically different negative DeltaCp values suggesting variable contribution from coupled protein folding and/or DNA structural transitions. We proposed that the CytR DNA binding-domain adopts either a more rigid or flexible DNA-bound conformation in response to the different spacer lengths. More recently, similar effects were shown to contribute to discrimination between operator and non-specific DNA binding by LacR, a CytR homolog. Here we have extended the thermodynamic analysis to the remaining natural CytR operators plus a set of synthetic operators designed to isolate spacing as the single variable. The thermodynamic results show a broad and monotonic range of effects that are primarily dependent on spacer length. The magnitude of effects suggests participation by more than the DNA-binding domain. 15N HSQC NMR and CD spectral analyses were employed to characterize the structural basis for these effects. The results indicate that while CytR forms a well-ordered structure in solution, it is highly dynamic. We propose a model in which a large ensemble of native state conformations narrows upon binding, to an extent governed by operator spacing. This in turn is expected to constrain intermolecular interactions in the CytR-CRP-DNA complex, thus generating operator-specific effects on repression and induction of transcription.

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