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

Genetics. 1999 May;152(1):129-41. doi: 10.1093/genetics/152.1.129.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Blotting, Northern
  • Cell Division
  • Cytochrome c Group / metabolism
  • Cytochromes c*
  • Genetic Complementation Test
  • Glycine-tRNA Ligase / genetics*
  • Models, Genetic
  • Mutagenesis
  • Plasmids
  • RNA, Transfer / metabolism
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae Proteins*
  • Sequence Analysis, DNA
  • Temperature
  • Transcription, Genetic* / physiology

Substances

  • CYC1 protein, S cerevisiae
  • Cytochrome c Group
  • Saccharomyces cerevisiae Proteins
  • Cytochromes c
  • RNA, Transfer
  • Glycine-tRNA Ligase