Figure 1.

Position-specific exclusion of sequence in RNase III substrates. (A) Substrate alignment analysis. Ten substrates were aligned whose cleavage sites were accurately determined, either by direct RNA sequence analysis or by primer extension of RNA cleaved in vitro by purified RNase III: 16S rRNA precursor (14), 23S rRNA precursor (15), T7 R0.3 (16), R0.5 (16), R1.1 (16) and R1.3 (16) substrates; lambda N leader (17), lambda sib (18) lambda cIII (19), and the RNase III operon transcript 5′-leader (20, 21). Each substrate provided two sequences for alignment, reflecting a two-fold symmetry about the cleavage site (11, 12). The cleavage site is indicated by vertical arrowhead between −1 and +1. The data represent the number of times (n = 20) each W-C bp occurs at positions +1 to −12. Position −12 represented the duplex limit for most substrates. P values from a Chi squared analysis also are provided. The significance of the disfavored A and U at positions −1 and −2, respectively, and the preferred C at −2 are not known, but may reflect specific sequence requirements for a structured asymmetric internal loop in a number of the substrates (22). The preference for CG at −6 is not known. (B) The “disfavored” bp, displayed in a dsRNA structure. The proximal box (PB) and distal box (DB) are included within an 11-bp helix. S = C or G, with S′ complementary to S. N, N′ indicate complementary nucleotides; while n, n′ indicate less strict complementarity. Arrowheads indicate the (blocked) cleavage sites. (C) Absence of conservation (degeneracy) of RNase III substrate sequence. H = A, G, U, with D′ (A, C, U) complementary to H; B = C, G, U, with V′ (G, C, U) complementary to B; W, W′ = A, U. (D) Secondary structure of the T7 R1.1 RNase III substrate, showing the proximal and distal boxes and the single cleavage site (arrowhead).