Ribosome Association of GCN2 Protein Kinase, a Translational Activator of the GCN4 Gene of Saccharomyces cerevisiae

Manuel Ramirez, Ronald Wek, Alan G. Hinnebusch

Research output: Contribution to journalArticle

104 Citations (Scopus)

Abstract

The GCN4 gene of the yeast Saccharomyces cerevisiae encodes a transcriptional activator of amino acid biosynthetic genes that is regulated at the translational level according to the availability of amino acids. GCN2 is a protein kinase required for increased translation of GCN4 mRNA in amino acid-starved cells. Centrifugation of cell extracts in sucrose gradients indicated that GCN2 comigrates with ribosomal subunits and polysomes. The fraction of GCN2 cosedimenting with polysomes was reduced under conditions in which polysomes were dissociated, suggesting that GCN2 is physically bound to these structures. When the association of 40S and 60S subunits was prevented by omitting Mg2+ from the gradient, almost all of the GCN2 comigrated with 60S ribosomal subunits, and it remained bound to these particles during gel electrophoresis under nondenaturing conditions. GCN2 could be dissociated from 60S subunits by 0.5 M KCl, suggesting that it is loosely associated with ribosomes rather than being an integral ribosomal protein. Accumulation of GCN2 on free 43S-48S particles and 60S subunits occurred during polysome runoff in vitro and under conditions of reduced growth rate in vivo. These observations, plus the fact that GCN2 shows preferential association with free ribosomal subunits during exponential growth, suggest that GCN2 interacts with ribosomes during the translation initiation cycle. The extreme carboxyl-terminal segment of GCN2 is essential for its interaction with ribosomes. These sequences are also required for the ability of GCN2 to stimulate GCN4 translation in vivo, leading us to propose that ribosome association by GCN2 is important for its access to substrates in the translational machinery or for detecting uncharged tRNA in amino acid-starved cells.

Original languageEnglish (US)
Pages (from-to)3027-3036
Number of pages10
JournalMolecular and Cellular Biology
Volume11
Issue number6
StatePublished - Jun 1991
Externally publishedYes

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Polyribosomes
Ribosomes
Protein Kinases
Saccharomyces cerevisiae
Ribosome Subunits
Amino Acids
Genes
Amino Acid-Specific Transfer RNA
Eukaryotic Large Ribosome Subunits
Free Association
Aptitude
Ribosomal Proteins
Protein Biosynthesis
Growth
Cell Extracts
Centrifugation
Sucrose
Electrophoresis
Yeasts
Gels

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

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Ribosome Association of GCN2 Protein Kinase, a Translational Activator of the GCN4 Gene of Saccharomyces cerevisiae. / Ramirez, Manuel; Wek, Ronald; Hinnebusch, Alan G.

In: Molecular and Cellular Biology, Vol. 11, No. 6, 06.1991, p. 3027-3036.

Research output: Contribution to journalArticle

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