SELECTIVELY C-13-ENRICHED DNA - EVIDENCE FROM CL-13' 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 C-13-labeled duplex 5'd(C(0)G(1)C(2)T(3)C(4)A(5)C(6)A(7) A(8)T(9)T(10)) d(A(10)A(9)T(8)T(7)G(6)T(5)G(4)A(3)G(2)C(1)G(0))31 was used. The spreading of both the H1' and Cl' resonances brought about a n excellent dispersion of the (1)Hl'-Cl-13' correlations. The spin-lat tice relaxation parameters R(C-z) R(C-x,C-y) and R(H-z-->C-z) were mea sured for each residue of the two complementary strands, except for th e 3'-terminal residues which were not labeled. Variation of the relaxa tion rates was found along the sequence. These data were analyzed in t he context of the model-free formalism proposed by Lipari and Szabo [( 1982) J. Am. Chem. Soc , 104, 4546-4570] and extended to three paramet ers by Clore et al. [(1990) Biochemistry, 29, 7387-7401; and (1990) J: Am. Chem. Sec, 112, 4989-4991]. A careful analysis using a least-squa res program showed that our data must be interpreted in terms of a thr ee-parameter spectral density function. With this approach, the global correlation time was found to be the same for each residue. All the C l'-Hl' fragments exhibited both slow (tau(s) = 1.5 ns) and fast (tau(f ) = 20 ps) restricted libration motions (S-s(2) = 0.74 to 1.0 and S-f( 2) = 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 b etween the amplitude motions of the complementary residues. The 5'-ter minal residues showed large internal motions (S-2 = 0.5), which descri be the fraying of the double helix. Global examination of the microdyn amical parameters S-f(2) and S-s(2) along the nucleotide sequence show ed that the adenine residues exhibit more restricted fast internal mot ions (S-f(2) = C).88 to 0.96) than the others, whereas the measured re laxation rates of the four nucleosides in solution were mainly of dipo lar origin. Moreover, the fit of both R(C-2) and R(Hz-->C-z) experimen tal relaxation rates using an only global correlation time for all the residues, gave evidence of a supplementary relaxation pathway affecti ng R(C-x,C-y) for the purine residues in the (5'-->3') G(4)A(3) and A( 10)A(9)T(3)T7 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 thes e residues gave evidence of close contacts with the protein in the com plex 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-l ac-repressor recognition process.