atpdependent interaction of yeast u5 snrna loop 1 with the 5 splice site premessenger rna splicing is a twostep process by which introns are removed and exons joined together in yeast the u5 snrna loop 1 interacts with the 5 exon before the first step of splicing and with the 5 and 3 exons before the second step in vitro studies revealed that yeast u5 loop 1 is not required for the first step of splicing but is essential for holding the 5 and 3 exons for ligation during the second step it is critical therefore that loop 1 contacts the 5 exon before the first step of splicing to hold this exon following cleavage from the premrna at present it is not known how u5 loop 1 is positioned on the 5 exon prior to the first step of splicing to address this question we have used sitespecific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of u5 loop 1 with the premrna prior to the first step of splicing we have found that the highly conserved uridines in loop 1 make atpdependent contacts with an approximately 8nt region at the 5 splice site that includes the invariant gu these interactions are dependent on functional u2 and u6 snrnas our results support a model where u5 snrna loop 1 interacts with the 5 exon in two steps during its targeting to the 5 splice site