A transcription terminator in the thymidylate synthase (thyA) structural gene of Escherichia coli and construction of a viable thyA::Kmr deletion.

A transcription terminator has been identified within the coding sequence of the Escherichia coli thyA gene. Fusion of a relevant segment of the thyA structural gene to galK sequences showed that the terminator functions in vivo. Primer extension and Northern hybridization (RNA blot) analysis of thyA RNA suggested that the terminator acts as the transcription stop signal for an upstream gene and for thyA-specific transcripts. Results from antitermination studies utilizing a lambda PL-thyA fusion also offer evidence that the terminator is capable of attenuating thyA expression by reducing the amount of full-length thyA transcripts. This gene arrangement suggested that previous unsuccessful attempts to create a chromosomal thyA deletion in E. coli were attributable to the presence of the overlapping transcript. Introducing a deletion into the nonoverlapping portion of the cloned thyA gene and inserting a gene encoding kanamycin resistance produced a (delta thyA::Kmr) that was easily transferred to the chromosome of a recD host by marker replacement. This delta thyA::Kmr allele provides a useful and readily transducible chromosomal marker.

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

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.