Domain 5 binds near a highly conserved dinucleotide in the joiner linking domains 2 and 3 of a group II intron

Abstract

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.