Defining the role of GRS1 in transcription termination
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Description
Eukaryotic mRNA 3' end formation is a complex process that involves termination of transcription and mRNA cleavage and polyadenylation, and these processes are tightly linked in vivo. Conflicting data in the literature concerning the mechanisms of polymerase release, transcript cleavage and subsequent termination indicate that there may be more than one mechanism directing RNA polymerase II termination. A screen designed to reveal trans-acting factors involved in transcription termination in Saccharomyces cerevisiae identified a temperature-sensitive mutant that displayed phenotypes consistent with a role in transcription termination. The complementing gene was identified as GRS1, which encodes the Saccharomyces cerevisiae glycyl tRNA synthetase. This result, although unusual, is not unprecedented given that tRNA synthetases are now known to be involved in a variety of cellular processes and bind substrates other than their cognate tRNAs. This dissertation utilized several in vitro and in vivo assays to explore the hypothesis that glycyl tRNA synthetase is directly involved in the termination process. The data show that the synthetase can interact directly and specifically with mRNA 3' ends in vitro and that it has a similar affinity for these substrates compared to tRNAgly. In addition, it was found that all mRNA and tRNA substrates compete with each other for binding to the synthetase. The effect of the grs1-1 mutation was also examined and found to significantly reduce the affinity of the enzyme for the three RNA substrates in vitro. Finally, versions of the mRNA 3' ends containing mutations resulting in deficient termination signals in vivo are unable to interact with the synthetase in vitro . Taken together, these data indicate that not only does this synthetase interact with several different RNA substrates, but also that there is a clear correlation between synthetase binding and termination efficiency. This provides support for the hypothesis that the Saccharomyces cerevisiae glycyl tRNA synthetase plays a direct role in transcription termination through an interaction with the 3' ends of mRNA. The identification of a synthetase as a transcription termination factor not only provides additional information into the complex termination process but also allows insight into the coordination of cellular events