Functional Interaction between Ribosomal Protein L6 and RbgA during Ribosome Assembly

Abstract

RbgA is an essential GTPase that participates in the assembly of the large ribosomal subunit in Bacillus subtilis and its homologs are implicated in mitochondrial and eukaryotic large subunit assembly. How RbgA functions in this process is still poorly understood. To gain insight into the function of RbgA we isolated suppressor mutations that partially restored the growth of an RbgA mutation (RbgA-F6A) that caused a severe growth defect. Analysis of these suppressors identified mutations in rplF, encoding ribosomal protein L6. The suppressor strains all accumulated a novel ribosome intermediate that migrates at 44S in sucrose gradients. All of the mutations cluster in a region of L6 that is in close contact with helix 97 of the 23S rRNA. In vitro maturation assays indicate that the L6 substitutions allow the defective RbgA-F6A protein to function more effectively in ribosome maturation. Our results suggest that RbgA functions to properly position L6 on the ribosome, prior to the incorporation of L16 and other late assembly proteins.

Author Summary

Ribosomes are complex macromolecular machines that carry out the essential function of protein synthesis in the cell. The assembly of ribosomal subunits is a multistep process that involves the accurate and timely assembly of 3 rRNA molecules and>50 ribosomal-proteins. In recent years many ribosome assembly factors have been identified in bacterial and eukaryotic cells; however, their precise functions in ribosome biogenesis are poorly understood. We have previously shown that the GTPase RbgA, a protein conserved from bacteria to humans, is essential for ribosome assembly in Bacillus subtilis. Here, we show that growth defect caused by a mutation in RbgA is partially suppressed by mutations in ribosomal protein L6. The suppressor strains accumulate novel ribosomal intermediates that appear to suppress the RbgA defect by weakening the interaction of L6 for the ribosome and facilitating RbgA dependent assembly. Our work provides evidence for a functional interaction between ribosome assembly factor RbgA and ribosomal protein L6 during assembly, a function that is likely important for mitochondrial, chloroplast, and eukaryotic ribosome assembly as well.