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One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs

Published online by Cambridge University Press:  20 August 2002

TOSHIFUMI INADA ,
ERIC WINSTALL ,
SALVADOR Z. TARUN ,
JOHN R. YATES ,
DAVE SCHIELTZ  and
ALAN B. SACHS
TOSHIFUMI INADA
Affiliation:
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA Present address: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan.
ERIC WINSTALL
Affiliation:
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA Present address: Centre Protéomique de l'est du Québec, Centre de recherche du CHUL, Université Laval, Québec, G1V 4G2, Canada.
SALVADOR Z. TARUN
Affiliation:
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA
JOHN R. YATES
Affiliation:
Department of Cell Biology SR11, The Scripps Research Institute, La Jolla, California 92037, USA
DAVE SCHIELTZ
Affiliation:
Department of Cell Biology SR11, The Scripps Research Institute, La Jolla, California 92037, USA
ALAN B. SACHS
Affiliation:
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA
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Abstract

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 starting 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.

Keywords

60S subunitFLAG tagpolysomeribosome-associated proteinRpl25ptranslation
Type
METHODS
Information
RNA , Volume 8 , Issue 7 , July 2002 , pp. 948 - 958
Copyright
2002 RNA Society

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