Transcription termination and 3' end formation are essential processes necessary for gene expression. However, the specific mechanisms responsible for these events remain elusive. A screen designed to identify trans-acting factors involved in these mechanisms 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 S. cerevisiae glycyl-tRNA synthetase. This result, although unusual, is not unprecedented given that the involvement of tRNA synthetases in a variety of cellular processes other than translation has been well established. A direct role for the synthetase in transcription termination was determined through several in vitro assays using purified wild type and mutant enzyme. First, binding to two well characterized yeast mRNA 3' ends was demonstrated by cross-linking studies. In addition, it was found that all three substrates compete with each other for binding to GlyRS enzyme. Next, the affinity of the synthetase for the two mRNA 3' ends was found to be similar to that of its "natural" substrate, glycine tRNA in a nitrocellulose filter binding assay. 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. Taken together, these data indicate that not only does this synthetase interact with several different RNA substrates, but also that these substrates bind to the same site. These results establish a direct role for GRS1 in mRNA 3' end formation.