Poster abstracts

Poster number 46 submitted by Anne Labut

Investigating the Regulation of Bms1, a GTPase Essential for 40S Ribosome Assembly

Anne E. Labut (Chemistry, University of Michigan), Katrin Karbstein (Chemistry, University of Michigan)

Abstract:
Ribosomes are responsible for catalyzing protein synthesis in all cells. Assembly of the four rRNAs and 78 ribosomal proteins in eukaryotic ribosomes requires a large molecular machinery of ~200 proteins and dozens of RNAs. GTPases are one class of assembly factors that is conserved from bacteria to humans and of specific interest due to its potential regulatory role. Previous work has demonstrated that the essential GTPase Bms1 promotes assembly of a pre-40S ribosomal particle essential for rRNA processing.¹ Here, we focus on the mechanism by which the GTPase activity within Bms1 is stimulated by an unusual intramolecular GTPase-activating (GAP) domain. We have devised an assay to identify the GAP domain in which GTPase and GAP domains are provided on separate molecules. This assay has identified a 150 amino acid region that provides GTPase stimulation in trans. Interestingly, this region is highly conserved including numerous essential amino acids as well as a conserved phosphorylation site, which regulates the activity of Bms1 in vivo. In vitro analysis of mutant Bms1 has identified two residues that impair GAP activation. The essential amino acids not involved in GTPase activation might be involved in regulating GTPase activity as a result of conformational rearrangements on pre-ribosomes as suggested by previous work.¹ Additionally, our experiments indicate the GAP domain is more effective when provided in trans than in cis, indicating the alignment of the domains in full length Bms1 protein is not optimized. We will continue to investigate the interaction of the GAP and GTPase domains in the presence and absence of pre-ribosomes and/or other accessory factors to determine whether these factors affect Bms1’s GAP-stimulated or basal GTPase activity.

References:
1. Karbstein, K., Jonas, S., Doudna, J. (2005) An Essential GTPase Promotes Assembly of Preribosomal RNA Processing Complexes. Mol. Cell 20, 633-643.

Keywords: ribosome assembly, small subunit, GTPase