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PMID: 12166649 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

RNA (New York, N.Y.) ·Vol. 8 ·No. 7 ·2002-07-00 ·Pages 948-58

Inada T, Winstall E, Tarun SZ, Yates JR, Schieltz D, Sachs AB

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 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.

MeSH Terms
Base Sequence Cell Fractionation/methods Chromatography, Affinity Plasmids/genetics RNA, Fungal/genetics,isolation & purification RNA, Messenger/genetics,isolation & purification Ribosomal Proteins/isolation & purification Ribosomes/chemistry Saccharomyces cerevisiae/chemistry,genetics Saccharomyces cerevisiae Proteins/isolation & purification
Chemicals
RNA, Fungal RNA, Messenger Ribosomal Proteins Saccharomyces cerevisiae Proteins
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Inada Toshifumi
Department of Molecular and Cell Biology, University of California at Berkeley, 94720, USA.
Winstall Eric
Tarun Salvador Z
Yates John R
Schieltz Dave
Sachs Alan B
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Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1355-8382
DOI
10.1017/s1355838202026018
Published
2002-07-00
Pages
948-58
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC1370311
Subset
IM
Grants
NIGMS NIH HHS · GM 50308 · United States
NCRR NIH HHS · RR11823 · United States
External Links
PubMed source DOI: 10.1017/s1355838202026018 PMC Full Text View journal details
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