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Molecular and Cellular Biology, March 2007, p. 2384-2397, Vol. 27, No. 6
0270-7306/07/$08.00+0     doi:10.1128/MCB.02254-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining{triangledown}

Jeanne M. Fringer,1 Michael G. Acker,2 Christie A. Fekete,1 Jon R. Lorsch,2 and Thomas E. Dever1*

Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892,1 Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 212052

Received 1 December 2006/ Returned for modification 26 December 2006/ Accepted 5 January 2007

The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.

* Corresponding author. Mailing address: NIH, Bldg. 6A/Rm. B1A-03, 6 Center Dr., Bethesda, MD 20892. Phone: (301) 496-4519. Fax: (301) 496-8576. E-mail: tdever{at}nih.gov.

{triangledown} Published ahead of print on 22 January 2007.

Molecular and Cellular Biology, March 2007, p. 2384-2397, Vol. 27, No. 6
0270-7306/07/$08.00+0     doi:10.1128/MCB.02254-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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