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.
Highlights
The assembly of the 30S and 50S ribosomal subunits is a complex and tightly coordinated series of events that consists of the synthesis, processing and modification of 5S, 16S and 23S rRNA and the addition of more than 50 ribosomal proteins (r-proteins) [1,2,3]
Fewer studies focused on ribosome assembly in other bacterial species, such as Geobacillus stearothermophilus, and these demonstrated that the intermediates formed in this system are different than those in E. coli, similar non-physiological steps are required for formation of a functional ribosomal subunit [5,8]
We have previously shown that the GTPase RbgA, a protein conserved from bacteria to humans, is essential for ribosome assembly in Bacillus subtilis
Summary
The assembly of the 30S and 50S ribosomal subunits is a complex and tightly coordinated series of events that consists of the synthesis, processing and modification of 5S, 16S and 23S rRNA and the addition of more than 50 ribosomal proteins (r-proteins) [1,2,3]. The in vitro reconstitution of a mature 50S subunit has been extensively studied in Escherichia coli and the formation of a mature 50S subunit from its constituent r-proteins and rRNA is a multi-step process that requires non-physiological conditions such as high ionic concentration, high temperatures and long incubation times [4,5,6,7]. Fewer studies focused on ribosome assembly in other bacterial species, such as Geobacillus stearothermophilus, and these demonstrated that the intermediates formed in this system are different than those in E. coli, similar non-physiological steps are required for formation of a functional ribosomal subunit [5,8]. The RbgA homolog Lsg has been proposed to play a role in the incorporation of the L16 homolog Rpl into the large ribosomal subunit, suggesting
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