MUTATIONAL ANALYSIS OF EXORIBONUCLEASE-I FROM SACCHAROMYCES-CEREVISIAE

Exoribonuclease I from yeast is a 175 kDa protein that is responsible for the majority of cytoplasmic mRNA degradation. Alignment of the Xrn 1p sequence with homologs from yeast as well as from higher eukaryotes suggests that the protein is composed of several domains: two acidic N-terminal domains which likely contain the exonuclease, a basic middl e domain and a basic C-terminal domain. Deletion analysis demonstrated that the C-terminus is dispensable for most in vivo and in vitro func tions but confers a dominant negative growth inhibition when expressed at high levels. This growth inhibition is not due to the exonuclease function of the protein. To identify specific residues responsible for in vivo function, a screen was carried out for non-complementing miss ense mutations. Fourteen single point mutations were identified that a ltered highly conserved amino acids within the first N-terminal domain of Xrn1p. All of the mutations reduced exonuclease activity measured in vivo and in vitro using affinity-purified proteins. The mutants fel l into two phenotypic classes, those that reduced or abolished exonucl ease activity without qualitatively changing the products of RNA degra dation and those that gave rise to novel degradation intermediates on certain RNAs.