The formation of the 3'-end of messenger RNA and termination of transcription by RNA polymerase II are events that are essential for gene expression; however, these processes have incompletely understood mechanisms that involve both cis-acting and trans -acting factors. As part of an effort to examine the mechanisms of transcription termination by RNA polymerase II, a large-scale genetic screen for strains defective in 3'-end formation was conducted. Following creation of a battery of mutant strains by 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 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 Once a temperature sensitive mutant strain with phenotypes consistent with a defect in transcription termination was identified, cloning by complementation of the temperature sensitivity 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 Several observations support the idea that the Grs1p is directly involved in 3' end formation, including that synthetases are involved in a variety of non-aminoacylation reactions, notably RNA splicing. Additionally, a suppressor of grs1-1, YMK1, was identified that is related to a previously identified 3'-end formation factors. Finally, structures with some similarity to tRNA molecules were observed within the 3'-end of various yeast genes. Based on these results, it is suggested that Grs1p is a transcription termination factor that interacts with the 3'-end of pre-mRNA to promote 3'-end formation / acase@tulane.edu
| Identifer | oai:union.ndltd.org:TULANE/oai:http://digitallibrary.tulane.edu/:tulane_27523 |
| Date | January 1999 |
| Contributors | Magrath, Christi Lee (Author), Hyman, Linda (Thesis advisor) |
| Publisher | Tulane University |
| Source Sets | Tulane University |
| Language | English |
| Detected Language | English |
| Rights | Access requires a license to the Dissertations and Theses (ProQuest) database., Copyright is in accordance with U.S. Copyright law |
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