rRNA Suppressor of a Eukaryotic Translation Initiation Factor 5B/Initiation Factor 2 Mutant Reveals a Binding Site for Translational GTPases on the Small Ribosomal Subunit

Abstract

The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit.

ACKNOWLEDGMENTS

We thank our colleagues in the Dever, Lorsch, and Hinnebusch laboratories for advice and helpful discussions; Jon Dinman for the PRF reporter plasmids; Preeti Saini for advice on the transit time measurements; and Alan Hinnebusch for comments on the manuscript.

This work was supported in part by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Development, National Institutes of Health (T.E.D.), and by American Cancer Society grant RSG-03-156-01-GMC (J.R.L.).