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Molecular and Cellular Biology, March 2007, p. 1677-1685, Vol. 27, No. 5
0270-7306/07/$08.00+0     doi:10.1128/MCB.01258-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Intragenic Suppressor Mutations Restore GTPase and Translation Functions of a Eukaryotic Initiation Factor 5B Switch II Mutant

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 11 July 2006/ Returned for modification 2 August 2006/ Accepted 11 December 2006

Structural studies of GTP-binding proteins identified the Switch I and Switch II elements as contacting the -phosphate of GTP and undergoing marked conformational changes upon GTP versus GDP binding. Movement of a universally conserved Gly at the N terminus of Switch II is thought to trigger the structural rearrangement of this element. Consistently, we found that mutation of this Gly in the Switch II element of the eukaryotic translation initiation factor 5B (eIF5B) from Saccharomyces cerevisiae impaired cell growth and the guanine nucleotide-binding, GTPase, and ribosomal subunit joining activities of eIF5B. In a screen for mutations that bypassed the critical requirement for this Switch II Gly in eIF5B, intragenic suppressors were identified in the Switch I element and at a residue in domain II of eIF5B that interacts with Switch II. The intragenic suppressors restored yeast cell growth and eIF5B nucleotide-binding, GTP hydrolysis, and subunit joining activities. We propose that the Switch II mutation distorts the geometry of the GTP-binding active site, impairing nucleotide binding and the eIF5B domain movements associated with GTP binding. Accordingly, the Switch I and domain II suppressor mutations induce Switch II to adopt a conformation favorable for nucleotide binding and hydrolysis and thereby reestablish coupling between GTP binding and eIF5B domain movements.

Published ahead of print on 22 December 2006.