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Development and characterization of a reconstituted yeast translation initiation system

Published online by Cambridge University Press:  24 April 2002

MIKKEL A. ALGIRE ,
DAVID MAAG ,
PETER SAVIO ,
MICHAEL G. ACKER ,
SALVADOR Z. TARUN ,
ALAN B. SACHS ,
KATSURA ASANO ,
KLAUS H. NIELSEN ,
DEANNE S. OLSEN  and
LON PHAN
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MIKKEL A. ALGIRE
Affiliation:
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
DAVID MAAG
Affiliation:
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
PETER SAVIO
Affiliation:
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
MICHAEL G. ACKER
Affiliation:
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
SALVADOR Z. TARUN
Affiliation:
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA
ALAN B. SACHS
Affiliation:
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA
KATSURA ASANO
Affiliation:
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA Present address: Division of Biology, Kansas State University, Manhattan, Kansas 66506, USA.
KLAUS H. NIELSEN
Affiliation:
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA
DEANNE S. OLSEN
Affiliation:
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA
LON PHAN
Affiliation:
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA Present address: National Center for Biotechnology Information/National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20892, USA.
ALAN G. HINNEBUSCH
Affiliation:
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA
JON R. LORSCH
Affiliation:
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
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Abstract

To provide a bridge between in vivo and in vitro studies of eukaryotic translation initiation, we have developed a reconstituted translation initiation system using components from the yeast Saccharomyces cerevisiae. We have purified a minimal set of initiation factors (eIFs) that, together with yeast 80S ribosomes, GTP, and initiator methionyl-tRNA, are sufficient to assemble active initiation complexes on a minimal mRNA template. The kinetics of various steps in the pathway of initiation complex assembly and the formation of the first peptide bond in vitro have been explored. The formation of active initiation complexes in this system is dependent on ribosomes, mRNA, Met-tRNAi, GTP hydrolysis, eIF1, eIF1A, eIF2, eIF5, and eIF5B. Our data indicate that eIF1 and eIF1A both facilitate the binding of the eIF2[bull]GTP[bull]Met-tRNAi complex to the 40S ribosomal subunit to form the 43S complex. eIF5 stimulates a step after 43S complex formation, consistent with its proposed role in activating GTP hydrolysis by eIF2 upon initiation codon recognition. The presence of eIF5B is required for the joining of the 40S and 60S subunits to form the 80S initiation complex. The step at which each of these factors acts in this reconstituted system is in agreement with previous data from in vivo studies and work using reconstituted mammalian systems, indicating that the system recapitulates fundamental events in translation initiation in eukaryotic cells. This system should allow us to couple powerful yeast genetic and molecular biological experiments with in vitro kinetic and biophysical experiments, yielding a better understanding of the molecular mechanics of this central, complex process.

Keywords

eIFeukaryotic initiation factorkineticsmechanismmRNAribosometRNA
Type
METHOD
Information
RNA , Volume 8 , Issue 3 , March 2002 , pp. 382 - 397
Copyright
© 2002 RNA Society

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