Obg is a GTP-binding protein of Bacillus subtilis with essential, but undefined roles in the bacterium's growth, sporulation, and stress responses. Obg orthologs are widely conserved among both bacteria and eukaryotes. Gel filtration and affinity blot assays have suggested that Obg may be ribosome-associated. In the current work, we continue an examination of the putative Obg:ribosome interaction. Velocity centrifugation analyses of crude B. subtilis extracts or purified Obg:ribosome mixtures suggest that Obg is initially ribosome-bound. but can separate from ribosomes during sedimentation in the absence of added nucleotides. Addition of either GTP, GDP or ATP to the gradient prolonged the Obg:ribosome association, while inclusion of a non hydrolyzable GTP analog (5-guanylyl-imidodiphosphate) preserved it. The data strengthen the notion that Obg is a ribosome-associated protein, demonstrate that Obg's association with ribosomes is stabilized by GTP, and indicate that the ribosome-bound Obg can likely hydrolyze GTP and be released as a consequence. (C) 2004 Elsevier Inc. All rights reserved.