Few point mutations have been described that specifically inhibit the second step of group II intron splicing. Furthermore, the effects of such mutations are typically not apparent unless the mutations are studied in the context of a substrate that harbors a very short 5′ exon. Truncation of the 5′ exon slows the second step of splicing. Once the second step has been slowed, the effects of point mutations can be seen. We report the unexpected observation that the deletion of a conserved GA dinucleotide dramatically inhibits the second step of splicing, even when the mutation is studied in the context of a full-length substrate. In contrast, we find that this mutation does not significantly affect the first step of splicing, unless the mutation is studied in combination with a second point mutation that is known to inhibit the first step. Even in that context, the effect of the GA deletion mutation on the first step is modest. These observations, together with the inferred location of the GA dinucleotide in the three-dimensional structure of the intron, suggest that this dinucleotide plays a particularly important role in the second step of splicing.

Dimethyl sulfate modification was used to probe for tertiary structural elements in the group II intron Pl.LSU/2 from the mitochondrial pre-ribosomal RNA of the brown alga Pylaiella littoralis. Modification of the lariat form of the intron under conditions that allow both native folding and conformational homogeneity is found to be generally consistent with secondary and tertiary structural features identified previously for group II ribozymes. A comparison of chemical probing at temperatures just below and above the first melting transition illustrates the cooperative unfolding of tertiary structure and identifies novel candidates for tertiary interactions in addition to defining elements of secondary structure. Substitution of the GAAA terminal loop of domain V is shown to be compatible with retention of conformational homogeneity (despite the loss of an important tertiary interaction), but produces a concise methylation footprint in domain I at the site previously shown to harbor the receptor for that loop. The analysis also identified two nucleotide positions in domain V with novel secondary and potential tertiary structural roles. The proposed refinement of domain V secondary structure is supported by an expanded comparative analysis of group II sequences and bears increased resemblance to U2:U6 snRNA pairing in the spliceosome.

Photocrosslinking has identified the joiner between domains 2 and 3 [J(23)] as folding near domain 5 (D5), a highly conserved helical substructure of group II introns required for both splicing reactions. D5 RNAs labeled with the photocrosslinker 4-thiouridine (4sU) reacted with highly conserved nucleotides G588 and A589 in J(23) of various intron acceptor transcripts. These conjugates retained some ribozyme function with the lower helix of D5 crosslinked to J(23), so they represent active complexes. One partner of the γ·γ′ tertiary interaction (A587·U887) is also in J(23); even though γ·γ′ is involved in step 2 of the splicing reaction, D5 has not previously been found to approach γ·γ′. Similar crosslinking patterns between D5 and J(23) were detected both before and after step 1 of the reaction, indicating that the lower helix of D5 is positioned similarly in both conformations of the active center. Our results suggest that the purine-rich J(23) strand is antiparallel to the D5 strand containing U32 and U33. Possibly, the interaction with J(23) helps position D5 correctly in the ribozyme active site; alternatively, J(23) itself might participate in the catalytic center.