One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs.
We describe a one-step affinity method for purifying ribosomes from the budding yeast Saccharomyces cerevisiae. Extracts from yeast strains expressing only C-terminally tagged Rpl25 protein or overexpressing this protein in the presence of endogenous Rpl25p were used as the starling materials. The purification was specific for tagged 60S subunits, and resulted in the copurification of 80S subunits and polysomes, as well as ribosome-associated proteins and mRNAs. Two of these associated proteins, Mpt4p and Asc1p, were nearly stoichiometrically bound to the ribosome. In addition, the degree of mRNA association with the purified ribosomes was found to reflect the mRNA's translational status within the cell. The one-step purification of ribosome and its associated components from a crude extract should provide an important tool for future structural and biochemical studies of the ribosome, as well as for expression profiling of translated mRNAs.
Inada
Toshifumi
T
Department of Molecular and Cell Biology, University of California at Berkeley, 94720, USA.
Winstall
Eric
E
Tarun
Salvador Z
SZ
Yates
John R
JR
Schieltz
Dave
D
Sachs
Alan B
AB
D001483:Base Sequence; D002458:Cell Fractionation / Q000379:methods; D002846:Chromatography, Affinity; D010957:Plasmids / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D012269:Ribosomal Proteins / Q000302:isolation & purification*; D012270:Ribosomes / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification*
D012331:RNA, Fungal; D012333:RNA, Messenger; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins