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.

[1]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[2]  J. Abelson,et al.  Isolation and sequence of the gene for actin in Saccharomyces cerevisiae. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[3]  J. Warner,et al.  The Yeast Ribosome: Structure, Function, and Synthesis , 1982 .

[4]  D. Luthe,et al.  A simple technique for the preparation and storage of sucrose gradients. , 1983, Analytical biochemistry.

[5]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[6]  A. Hinnebusch.  Evidence for translational regulation of the activator of general amino acid control in yeast. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[7]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[8]  J. Warner,et al.  10 The Ribosome and Its Synthesis , 1991 .

[9]  D. Tollervey,et al.  Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly , 1993, Cell.

[10]  Gerald R. Fink,et al.  Guide to yeast genetics and molecular biology , 1993 .

[11]  J. McCarthy,et al.  Inhibition of translational initiation in Saccharomyces cerevisiae by secondary structure: the roles of the stability and position of stem‐loops in the mRNA leader , 1993, Molecular microbiology.

[12]  R. Müller,et al.  Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. , 1994, Nucleic acids research.

[13]  W. Gilbert,et al.  A Yeast Gene Product, G4p2, with a Specific Affinity for Quadruplex Nucleic Acids (*) , 1995, The Journal of Biological Chemistry.

[14]  Wei Zhou,et al.  Characterization of the Yeast Transcriptome , 1997, Cell.

[15]  C. Lions,et al.  The transcriptional regulator Hap1p (Cyp1p) is essential for anaerobic or heme-deficient growth of Saccharomyces cerevisiae: Genetic and molecular characterization of an extragenic suppressor that encodes a WD repeat protein. , 1998, Genetics.

[16]  H. Mitsui,et al.  Dhh1p, a putative RNA helicase, associates with the general transcription factors Pop2p and Ccr4p from Saccharomyces cerevisiae. , 1998, Genetics.

[17]  P. Brown,et al.  Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[18]  J. Yates,et al.  Direct analysis of protein complexes using mass spectrometry , 1999, Nature Biotechnology.

[19]  D. Tollervey,et al.  A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants , 1999, The Journal of cell biology.

[20]  M. Schummer,et al.  Messenger RNA translation state: the second dimension of high-throughput expression screening. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Kenneth M. Kuhn,et al.  19 Cellular Internal Ribosome Entry Site Elements and the Use of cDNA Microarrays in Their Investigation , 2000 .

[22]  R. Parker,et al.  Three conserved members of the RNase D family have unique and overlapping functions in the processing of 5S, 5.8S, U4, U5, RNase MRP and RNase P RNAs in yeast , 2000, The EMBO journal.

[23]  John J. Wyrick,et al.  Genome-wide location and function of DNA binding proteins. , 2000, Science.

[24]  T. Steitz,et al.  The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. , 2000, Science.

[25]  A. Sachs,et al.  Glucose depletion rapidly inhibits translation initiation in yeast. , 2000, Molecular biology of the cell.

[26]  S. Tenenbaum,et al.  Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[27]  J. Shabanowitz,et al.  Composition and functional characterization of yeast 66S ribosome assembly intermediates. , 2001, Molecular cell.

[28]  J. Darnell,et al.  Microarray Identification of FMRP-Associated Brain mRNAs and Altered mRNA Translational Profiles in Fragile X Syndrome , 2001, Cell.

[29]  A. Ohta,et al.  Ray38p, a homolog of a purine motif triple-helical DNA-binding protein, Stm1p, is a ribosome-associated protein and dissociated from ribosomes prior to the induction of cycloheximide resistance in Candida maltosa. , 2001, Biochemical and biophysical research communications.

[30]  H. Beug,et al.  Translation control: bridging the gap between genomics and proteomics? , 2001, Trends in biochemical sciences.

[31]  Patrick O. Brown,et al.  Global and Specific Translational Regulation in the Genomic Response of Saccharomyces cerevisiae to a Rapid Transfer from a Fermentable to a Nonfermentable Carbon Source , 2001, Molecular and Cellular Biology.

[32]  P. Grandi,et al.  Identification of a 60S preribosomal particle that is closely linked to nuclear export. , 2001, Molecular cell.

[33]  J. Darnell,et al.  Fragile X Mental Retardation Protein Targets G Quartet mRNAs Important for Neuronal Function , 2001, Cell.