Selectively 13C-enriched DNA: evidence from 13C1' relaxation rate measurements of an internal dynamics sequence effect in the lac operator.
In order to study some internal dynamic processes of the lac operator sequence, the 13C-labeled duplex 5'd(C0G1C2T3C4A5C6A7A8T9T10).d(A10A9T8T7G6T5G4A3G2C1G0)3' was used. The spreading of both the 1H1' and 13C1' resonances brought about an excellent dispersion of the 1H1'-13C1' correlations. The spin-lattice relaxation parameters R(Cz), R(Cx,y) and R(Hz --> Cz) were measured for each residue of the two complementary strands, except for the 3'-terminal residues which were not labeled. Variation of the relaxation rates was found along the sequence. These data were analyzed in the context of the model-free formalism proposed by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546-4570] and extended to three parameters by Clore et al. [(1990) Biochemistry, 29, 7387-7401; and (1990) J. Am. Chem. Soc., 112, 4989-4991]. A careful analysis using a least-squares program showed that our data must be interpreted in terms of a three-parameter spectral density function. With this approach, the global correlation time was found to be the same for each residue. All the C1'-H1' fragments exhibited both slow (tau s = 1.5 ns) and fast (tau f = 20 ps) restricted libration motions (Ss2 = 0.74 to 1.0 and Sf2 = 0.52 to 0.96). Relaxation processes were described as governed by the motion of the sugar relative to the base and in terms of bending of the whole duplex. The possible role played by the special structure of the AATT sequence is discussed. No evident correlation was found between the amplitude motions of the complementary residues. The 5'-terminal residues showed large internal motions (S2 = 0.5), which describe the fraying of the double helix. Global examination of the microdynamical parameters Sf2 and Ss2 along the nucleotide sequence showed that the adenine residues exhibit more restricted fast internal motions (Sf2 = 0.88 to 0.96) than the others, whereas the measured relaxation rates of the four nucleosides in solution were mainly of dipolar origin. Moreover, the fit of both R(Cz) and R(Hz --> Cz) experimental relaxation rates using an only global correlation time for all the residues, gave evidence of a supplementary relaxation pathway affecting R(Cx,y) for the purine residues in the (5' --> 3') G4A3 and A10A9T8T7 sequences. This relaxation process was analyzed in terms of exchange stemming from motions of the sugar around the glycosidic bond on the millisecond time scale. It should be pointed out that these residues gave evidence of close contacts with the protein in the complex with the lac operator [Boelens et al. (1987) J. Mol. Biol., 193, 213-216] and that these motions could be implied in the lac-operator-lac-repressor recognition process.
Paquet
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F
Centre de Biophysique Moléculaire, CNRS, Orléans, France.
Gaudin
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F
Lancelot
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G
D001426:Bacterial Proteins / Q000235:genetics*; D002250:Carbon Radioisotopes; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D029968:Escherichia coli Proteins; D007763:Lac Operon / Q000235:genetics*; D056968:Lac Repressors; D009682:Magnetic Resonance Spectroscopy; D012097:Repressor Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012097:Repressor Proteins; D004247:DNA