Efficient control of group I intron ribozyme catalysis by DNA constraints

Abstract

Double-stranded DNA constraints enable efficient control of catalysis by a large multi-domain group I intron ribozyme.

Fig. 1

General DNA constraint strategy to control RNA conformation.,,

Fig. 2

Structure of the Tetrahymena group I intron ribozyme with DNA attachment sites. Fig. S1 in the ESI shows the secondary structure of the 388 nt L—21 ScaI ribozyme form of the intron RNA., Depicted here is the 3.8 Å X-ray crystal structure obtained with a truncated version of the ribozyme that lacks the P1 and P2 regions., The solid lines connecting 2′-OH groups (spheres) denote the seven pairwise combinations of attachment sites used to introduce DNA constraints.

Fig. 3

Pathways for group I intron folding. See text for explanation.

Fig. 4

Suppression of group I intron ribozyme catalysis by DNA constraints at A146-A97, A146-A286, or A146-A308 (single-turnover substrate cleavage assays, 100 mM HEPES, pH 7.2, 10 mM Mg²⁺, 100 mM NaCl, and 1 mM GTP at 30 °C). The 20% PAGE images show 10 min timepoints for cleavage of the 5′-³²P-radiolabeled 11-mer RNA substrate by a DNA-constrained ribozyme (Fig. 2, orange lines). The RNA substrate (filled arrowhead) is cleaved after U6 to form the product (open arrowhead). The attached DNA strands were either absent (-), complementary (compl), or noncomplementary (nc). The free oligonucleotide complementary to one of the two constraint strands was added for the lanes marked (+). The single-exponential kinetic fits are intended solely to guide the eye; the rates are too high under these incubation conditions to obtain precise k_obs values. Filled symbols indicate complementary DNA strands attached to the ribozyme; open symbols indicate complementary DNA strands after adding free oligonucleotide to release the constraint; crosses indicated noncomplementary DNA strands.