C Magrath | PubMed Health

PubMed1999

A mutation in GRS1, a glycyl-tRNA synthetase, affects 3'-end formation in Saccharomyces cerevisiae.

3'-end formation is a complex and incompletely understood process involving both cis-acting and trans-acting factors. As part of an effort to examine the mechanisms of transcription termination by RNA polymerase II, a mutant hunt for strains defective in 3'-end formation was conducted. Following random mutagenesis, a temperature-sensitive strain exhibiting several phenotypes consistent with a role in transcription termination was isolated. First, readthrough of a terminator increases significantly in the mutant strain. Accordingly, RNA analysis indicates a decrease in the level of terminated transcripts, both in vivo and in vitro. Moreover, a plasmid stability assay in which high levels of readthrough lead to high levels of plasmid loss and transcription run-on analysis also demonstrate defective termination of transcription. Examination of polyadenylation and cleavage by the mutant strain indicates these processes are not affected. These results represent the first example of a transcription termination factor in Saccharomyces cerevisiae that affects transcription termination independent of 3'-end processing of mRNA. Complementation studies identified GRS1, an aminoacyl-tRNA synthetase, as the complementing gene. Sequence analysis of grs1-1 in the mutant strain revealed that nucleotides 1656 and 1657 were both C to T transitions, resulting in a single amino acid change of proline to phenylalanine. Further studies revealed GRS1 is essential, and the grs1-1 allele confers the temperature-sensitive growth defect associated with the mutant strain. Finally, we observed structures with some similarity to tRNA molecules within the 3'-end of various yeast genes. On the basis of our results, we suggest Grs1p is a transcription termination factor that may interact with the 3'-end of pre-mRNA to promote 3'-end formation.

Blotting, Northern↗

PubMed1998

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.

Base Sequence↗