Nuclear magnetic resonance study of the proton exchange rate in the operator-promoter DNA sequence of the trp operon of Escherichia coli

Jean François Lefevre, Andrew N. Lane, Oleg Jardetzky

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27 Scopus citations


The dynamic behavior of a palindromic oligonucleotide (C-G-T-A-C-T-A-G-T-T-A-A-C-T-A-G-T-A-C-G) representative of the operator sequence and containing the Pribnow box of the trp operon of Escherichia coli was investigated. The resonances of the imino protons and of the H2 protons of the adenosine residues were all assigned. The opening rate constants of the base-pairs were calculated by monitoring the exchange rate of the observable imino protons (nine out of ten), using selective temperature (T1) measurements, which avoid the complication of cross-relaxation and spin diffusion. These measurements have to be performed in conditions where the exchange process is much faster than the opening and closing of the base-pairs, so that the observed exchange rate is equal to the opening rate. It is shown that the catalysis of the exchange process by phosphate dianions is not very efficient (kB ≈ 7 × 104 M-1 S-1). Hence, in phosphate buffer, the necessary opening-rate limiting condition is met only at high pH values (≈9.5) where efficient catalysis by OH- takes place, or at very high buffer concentration. While G · C base-pairs show very little exchange, acting in the sequence as molecular "staples", the A · T base-pairs that are protected from the fraying have very different opening and closing rates, depending on the sequence. Although it seems possible that the opening process could occur at the base-pair level, it is localized at most to two base-pairs in that particular sequence. The activation energies for the opening process of all non-fraying base-pairs are very similar (19 ± 1 kcal mol-1; 1 cal = 4.184 J), and the differences in the opening rates are essentially due to differences in the activation entropies. With regard to the role of this sequence in the promoter, it is observed that the end of the Pribnow box exchanges relatively easily, and that the activation parameters for the "breathing" process and for the isomerization step of the promoter-RNA polymerase are not very different.

Original languageEnglish
Pages (from-to)689-699
Number of pages11
JournalJournal of Molecular Biology
Issue number4
StatePublished - Oct 20 1985

Bibliographical note

Funding Information:
This research was supported by NIH grant RROO711. \Ve thank EMBO for a long-termf ellowship (to A.X.L.). h’AT0 and the Philippe Foundation for support (to .J.F.L.).

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


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