Structure and Conformation of the Duplex Consensus 5′-Splice Site d [(CpApGpGpTpApApGpT) (ApCpTpTpApCpCpTpG)] Deduced from High Field1H-NMR of the Non-Exchangeable and Imino Protons

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Abstract
The complementary consensus donor exon:intron junction d(ApCpTpTpApCpCpTpG) has been synthesized by a solid phase procedure. The non-exchangeable proton assignments were obtained using one- and two-dimensional NMR techniques including NOE, COSY, NOESY and 1H-1H-INADEQUATE. The non self-complementary nonamer exists as a random coil form in aqueous buffer at 21 °C as evidenced by the temperature variable 1H-NMR and NOE measurements. The nonamer was annealed to the primary consensus donor junction d(CpApGpGpTpApApGpT) and confirmation of complete annealing was obtained by detection and assignment of base pair imino protons in D2O/H2O mixtures. Application of one- and two-dimensional NMR techniques permitted the complete assignment of all the non-exchangeable protons in the duplex nonamer. These data, together with determination of vicinal coupling constants in the individual deoxyribose moieties, permits the following conclusions on the structure and conformation of the consensus donor junction: (i) it exists in aqueous solution in a conformation that belongs to the B family (ii) the sugar-base orientations are anti (iii) the deoxyribose units exist predominantly in the S conformation (2′-endo-3′-exo) (iv) the contiguous A · Tbase pairs d[T(5)-A(6)-A(7)] · d[T(12)-T(13)-A(14)], two positions removed downstream from the splice site (5′-C AG|GTAAGT-3′), are uniquely propeller twisted. The propeller twisting occurs in the region in which there is partial complementarity with the branch site splice signal TACTAAC. The cross-correlation rates were used to derive the interproton distances between adjacent AH2 protons of 4.00 Å in the T(5)—A(6) · T(13)—A(14) step and of 3.87 Å in the A(6)—A(7) · T(12)—T(13) step. This structural and conformational feature if carried over into the primary RNA transcripts may serve as a recognition signal for this critical site in the genome.