DOMAIN-5 BINDS NEAR A HIGHLY CONSERVED DINUCLEOTIDE IN THE JOINER LINKING DOMAIN-2 AND DOMAIN-3 OF A GROUP-II INTRON

Photocrosslinking has identified the joiner between domains 2 and 3 [J (23)] as folding near domain 5 (D5), a highly conserved helical substr ucture of group II introns required for both splicing reactions. D5 RN As labeled with the photocrosslinker 4-thiouridine (4sU) reacted with highly conserved nucleotides G(588) and A(589) in J(23) of various int ron acceptor transcripts. These conjugates retained some ribozyme func tion with the lower helix of D5 crosslinked to 5(23), so they represen t active complexes. One partner of the gamma.gamma' tertiary interacti on (A(587).U-887) is also in 5(23); even though gamma.gamma' is involv ed in step 2 of the splicing reaction, D5 has not previously been foun d to approach gamma.gamma'. Similar crosslinking patterns between D5 a nd J(23) were detected both before and after step 1 of the reaction, i ndicating that the lower helix of D5 is positioned similarly in both c onformations of the active center. Our results suggest that the purine -rich J(23) strand is antiparallel to the D5 strand containing U-32 an d U-33. Possibly, the interaction with J(23) helps position D5 correct ly in the ribozyme active site; alternatively, J(23) itself might part icipate in the catalytic center.