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Cited by 129RNA and protein synthesis mechanismsRNA Research and SplicingGenomics and Chromatin Dynamics

Dissecting eukaryotic translation and its control by ribosome density mapping

Open access
Arava, Y.·2005·Nucleic Acids Research
DOI 10.1093/nar/gki331PMC PMC1087779
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Abstract

Translation of an mRNA is generally divided into three stages: initiation, elongation and termination. The relative rates of these steps determine both the number and position of ribosomes along the mRNA, but traditional velocity sedimentation assays for the translational status of mRNA determine only the number of bound ribosomes. We developed a procedure, termed Ribosome Density Mapping (RDM), that uses site-specific cleavage of polysomal mRNA followed by separation on a sucrose gradient and northern analysis, to determine the number of ribosomes associated with specified portions of a particular mRNA. This procedure allows us to test models for translation and its control, and to examine properties of individual steps of translation in vivo. We tested specific predictions from the current model for translational control of GCN4 expression in yeast and found that ribosomes were differentially associated with the uORFs elements and coding region under different growth conditions, consistent with this model. We also mapped ribosome density along the ORF of several mRNAs, to probe basic kinetic properties of translational steps in yeast. We found no detectable decline in ribosome density between the 5' and 3' ends of the ORFs, suggesting that the average processivity of elongation is very high. Conversely, there was no queue of ribosomes at the termination site, suggesting that termination is not very slow relative to elongation and initiation. Finally, the RDM results suggest that less frequent initiation of translation on mRNAs with longer ORFs is responsible for the inverse correlation between ORF length and ribosomal density that we observed in a global analysis of translation. These results provide new insights into eukaryotic translation in vivo.

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  • The estimated rates provide an average over the population and time and do not describe gene expression in single cells.

    “The experimental data is based on a population of non-synchronized cells. Therefore, our estimated rates provide an average over the population and time. They do not describe gene expression in single cells which requires single cell measurements 26 .”

    From Global quantification of mammalian gene expression control (2011) · cites this paper as [26]

    The Results and Discussion section (‘Exploring the stages of translation of yeast mRNAs by RDM’; page unavailable) describes isolating polysomes from yeast cells and analyzing fractions by northern blot, supporting the distinction from single-cell measurements. The paper does not say that the citing authors’ estimated rates average over time or over a non-synchronized population. (LLM, conf=0.78)

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Research works printed but not linked: 8

  1. [1]Rickwood D. Preparative Centrifugation. A Practical Approach, 2nd edn. 1992 Oxford, New York
  2. [14]MacKay V.L. Li X. Flory M.R. Turcott E. Law G.L. Serikawa K.A. Xu X.L. Lee H. Goodlett D.R. Aebersold R. Gene expression analyzed by high-resolution state array analysis and quantitative proteomics: response of yeast to mating pheromone Mol. Cell Proteomics 2004 3 478 489 14766929
  3. [25]Yaman I. Fernandez J. Liu H. Caprara M. Komar A.A. Koromilas A.E. Zhou L. Snider M.D. Scheuner D. Kaufman R.J. The zipper model of translational control: a small upstream ORF is the switch that controls structural remodeling of an mRNA leader Cell 2003 113 519 531 12757712
  4. [29]Hinnebusch A.G. Translational regulation of yeast GCN4. A window on factors that control initiator-tRNA binding to the ribosome J. Biol. Chem. 1997 272 21661 21664 9268289
  5. [31]Geballe A.P. Sachs M.S. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational control by upstream open reading frame Translational Control of Gene Expression 2000 Cold Spring Harbor CSHL Press
  6. [34]Tsung K. Inouye S. Inouye M. Factors affecting the efficiency of protein synthesis in Escherichia coli. Production of a polypeptide of more than 6000 amino acid residues J. Biol. Chem. 1989 264 4428 4433 2538444
  7. [36]Farabaugh P.J. Qian Q. Stahl G. Sonenberg N. Hershey J.W.B. Mathews M.B. Programmed translational frameshifting, hopping and readthrough of termination codons Translational Control of Gene Expression 2000 Cold Spring Harbor CSHL Press
  8. [38]Sachs A.B. Sonenberg N. Hershey J.W.B. Mathews M.B. Physical and functional interactions between the mRNA cap structure and the polyA tail Translational Control of Gene Expression 2000 Cold Spring Harbor CSHL Press

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Authors
Arava, Y.
Affiliations
Howard Hughes Medical Institute (US)
Corresponding author
Arava, Y.
ORCID
Arava, Y.
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2005-04-28
Volume
33
Issue
8
Pages
2421-2432
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gki331
PMC
PMC1087779
OpenAlex
W2133163771
Cited by
129
References
32
Citations by year
2026: 1 · 2023: 3 · 2022: 3 · 2021: 5 · 2020: 3 · 2019: 3 · 2018: 4 · 2017: 9 · 2016: 4 · 2015: 9 · 2014: 8 · 2013: 11 · 2012: 8
FWCI
2.39
Type
journal article
Language
en
Open access
Licence
other-oa
Locations
  • Nucleic Acids Research (publishedVersion, other-oa)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Howard Hughes Medical Institute
  • American Heart Association
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords
eukaryotic translation, polysomal mRNA, translation initiation, translation elongation, translation termination, GCN4, upstream open reading frames, yeast, translational regulation, site-specific cleavage
MeSH terms
DNA-Binding Proteins, Genetic Techniques, Models, Genetic, Peptide Chain Elongation, Translational, Peptide Chain Initiation, Translational, Peptide Chain Termination, Translational, Protein Kinases, Ribosomes, RNA, Messenger, Saccharomyces cerevisiae, Protein Biosynthesis, Gene Expression Regulation, Fungal, Open Reading Frames, Saccharomyces cerevisiae Proteins

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