Overlapping 3'-end formation signals and ARS elements: tightly linked but functionally separable

3'-End formation signals are closely associated with autonomous replicating sequences (ARSs) in Saccharomyces cerevisiae in that ARSs frequently contain signals that direct 3'-end formation (Chen et al., 1996). Mutationally-inactivated ARSs that co-reside with 3'-end formation sequences do not disrupt 3'-end formation, thus demonstrating that replication function does not affect termination function. To test the corollary possibility that 3'-end formation is important for replication function, we made point mutations in ARS305 that increase readthrough of the 3'-end formation signals and determined plasmid replication efficiency. Replication efficiency, as assessed by plasmid stability assays, was not altered by mutations affecting 3'-end formation when transcription through the ARS was either absent or highly-induced. Under conditions of high-level transcription through the ARS, the rate of plasmid loss in both wild-type and mutated terminators increased over five-fold from rates observed during transcriptionally repressed conditions. This result indicates that the native 3'-end formation signal is incapable of protecting the replication function when high levels of transcription are directed into the ARS. Thus, the compact nature of the S. cerevisiae genome, rather than a functional inter-dependence, may account for close association of transcription terminators and ARSs.