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 Xrn1p 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 middle domainand a basic C-terminal domain. Deletion analysisdemonstrated that the C-terminus is dispensable for most in vivo and in vitro functions 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 missense mutations. Fourteen single point mutations were identified that altered 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 fell into two phenotypic classes, those that reduced or abolished exonuclease activity without qualitatively changing the products of RNA degradation and those that gave rise to novel degradation intermediates on certain RNAs.
Department of Microbiology and the Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712-1095, USA.
A M
AM
Page
K
K
Davis
C
C
Molineux
R D
RD
Kolodner
A W
AW
Johnson
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000235:genetics; D005095:Exoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes / Q000235:genetics; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D012331:RNA, Fungal / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid
D004271:DNA, Fungal; D015345:Oligonucleotide Probes; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D005095:Exoribonucleases; C068398:XRN1 protein, S cerevisiae