Abstract
Selection of the nucleophile for the first step of nuclear pre-mRNA splicing was probed by site-specific incorporation into splicing substrates of nucleotides modified at the 2' position. The differing abilities of ribose, 2'-deoxyribose, and arabinose nucleotides to base-pair within an RNA.RNA duplex and to contribute a nucleophilic 2'-OH group were exploited to analyze the paired/unpaired disposition of the branch site nucleotide. The results provide direct evidence for a bulged duplex model in which either of two adjacent purines within the consensus branch site sequence may shift into a bulged position and contribute the 2'-OH group for the first step of splicing. Furthermore, the presence of a consensus branch site that cannot present a reactive nucleophile suppresses splicing, including the use of cryptic branch sites elsewhere. We conclude that the branch site region base-pairing with U2 snRNA determines the first step nucleophile and persists at the time of the first transesterification reaction.
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