Poster abstracts
Poster number 24 submitted by Michelle Gibbs
LepA functions in assembly of the 30S subunit of the ribosome
Michelle R. Gibbs (Department of Microbiology and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Kyung-Mee Moon (Centre for High-Throughput Biology and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada), Menglin Chen (Ohio State Biochemistry Program, Department of Microbiology and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Rohan Balakrishnan (Ohio State Biochemistry Program, Department of Microbiology and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Leonard J. Foster (Centre for High-Throughput Biology and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada), Kurt Fredrick (Ohio State Biochemistry Program, Department of Microbiology and Center for RNA Biology, The Ohio State University, Columbus, OH 43210)
Abstract:
The GTPase LepA is a paralog of elongation factor EF-G found in all bacteria. The physiological role of LepA has remained elusive for decades. A popular model was that LepA promotes “back-translocation”, the reverse movement of tRNA-mRNA in the ribosome (Qin et al., 2006). Our laboratory and others have been unable to confirm that LepA has such activity. Instead, we found that LepA contributes mainly to translation initiation, either directly or indirectly (Balakrishnan et al., 2014). One hypothesis is that LepA participates in ribosome biogenesis and immature subunits formed in its absence are responsible for the observed initiation effects. To investigate this hypothesis, we used SILAC (stable isotope labeling of amino acids in culture) and mass spectrometry in this study to determine r-protein composition of ribosomal particles in the presence and absence of LepA. We find that four r-proteins are disproportionately underrepresented in 30S particles in the ΔlepA strain compared to the wild-type. These r-proteins (S3, S10, S14, S21) are all late-stage assembly proteins and localize to the 3’ domain of the 16S rRNA. In addition, we find that 30S and 70S particles in the ΔlepA mutant contain higher proportions of 17S precursor rRNA than in the wild-type. Collectively, these data indicate that LepA functions in 30S subunit assembly. SILAC analysis of r-protein composition in ΔrsgA cells showed a similar defect in 30S subunit assembly. RsgA is another GTPase known to be involved in 30S subunit biogenesis which was previously linked to LepA through a synthetic phenotype screen (Balakrishnan et al., 2014). Hence, LepA and RsgA appear to play partially redundant roles in 30S subunit assembly.
References:
Qin Y., Polacek N., Vesper O., Staub E., Einfeldt E., Wilson D.N., Nierhaus K.H. The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome. Cell. 2006;127:721–733.
Balakrishnan R, Oman K, Shoji S, Bundschuh R, Fredrick K. The conserved GTPase LepA contributes mainly to translation initiation in Escherichia coli. Nucleic Acids Res. 2014;42:13370–13383.
Keywords: LepA, RsgA, 30S ribosome assembly