Deletion-tolerance and trans-splicing of the bacteriophage T4 td intron. Analysis of the P6-L6a region

Abstract

Non-directed mutagenesis and phylogenetic comparison suggest that certain elements of the bacteriophage T4 td group Ia intron are dispensable to self-splicing. The L6-P6a-L6a region was identified as a potential non-essential element, and was removed by sequential deletions extending from the L6a loop toward the P6 pairing. Assays for splicing indicate that as long as the P6 pairing is maintained, the 1016 nucleotide td intron can be reduced to less than 250 nucleotides while maintaining function in vivo and in vitro. The P6 pairing appears to be essential for splicing while P6a is not. In addition, a spontaneous pseudorevertant of a splicing-defective deletion was isolated and shown to result from a single nucleotide change in the predicted L6a loop. This genetic suppressor mimics the ability of Mg2+ to reverse the phenotype of the deletion, suggesting that function is restored by structural stabilization of P6. The tolerance of this region to deletion prompted us to split the ribozyme core in L6a, to generate precursors that might function in trans. Indeed, the two half-molecules do associate to form a bimolecular complex that yields accurately ligated exons both in vitro and in vivo. The biological implications of these results, as well as the usefulness of trans-splicing for generating unprocessed precursors in vitro are discussed.