Tight binding of the 5 ' exon to domain I of a group II self-splicing intron requires completion of the intron active site

Group II self-splicing requires the 5' exon to form base pairs with two str etches of intronic sequence (EBS1 and EBS2) which also bind the DNA target during retrotransposition of the intron. We have used dimethyl sulfate modi fication of bases to obtain footprints of the 5' exon on intron Pl.LSU/2 fr om the mitochondrion of the alga Pylaiella littoralis, as well as on trunca ted intron derivatives. Aside from the EBS sites, which are part of the sam e subdomain (ID) of ribozyme secondary structure, three distant adenines be come either less or more sensitive to modification in the presence of the e xon, Unexpectedly, one of these adenines in subdomain IC1 is footprinted on ly in the presence of the distal helix of domain V, which is involved in ca talysis, While the loss of that footprint is accompanied by a 100-fold decr ease in the affinity for the exon, both protection from modification and ef ficient binding can be restored by a separate domain V transcript, whose bi nding results in its own, concise footprint on domains I and III. Possible biological implications of the need for the group II active site to be comp lete in order to observe high-affinity binding of the 5' exon to domain I a re discussed.