RNA



METHODS
METHODS

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


TOSHIFUMI  INADA  a1 p1, ERIC  WINSTALL  a1 p2, SALVADOR Z.  TARUN  JR. a1, JOHN R.  YATES  III a2, DAVE  SCHIELTZ  a2 and ALAN B.  SACHS  a1 c1
a1 Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA
a2 Department of Cell Biology SR11, The Scripps Research Institute, La Jolla, California 92037, USA

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.

(Received March 26 2002)
(Revised April 5 2002)
(Accepted April 26 2002)


Key Words: 60S subunit; FLAG tag; polysome; ribosome-associated protein; Rpl25p; translation.

Correspondence:
c1 Reprint requests to: Alan B. Sachs, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA; e-mail: asachs@uclink4.berkeley.edu.
p1 Present address: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan.
p2 Present address: Centre Protéomique de l'est du Québec, Centre de recherche du CHUL, Université Laval, Québec, G1V 4G2, Canada.