Defect in the Formation of 70S Ribosomes Caused by Lack of Ribosomal Protein L34 Can Be Suppressed by Magnesium

Abstract

dTo elucidate the biological functions of the ribosomal protein L34, which is encoded by the rpmH gene, the rpmH deletion mutant of Bacillus subtilis and two suppressor mutants were characterized. Although the ⌬rpmH mutant exhibited a severe slowgrowth phenotype, additional mutations in the yhdP or mgtE gene restored the growth rate of the ⌬rpmH strain. Either the disruption of yhdP, which is thought to be involved in the efflux of Mg 2؉ , or overexpression of mgtE, which plays a major role in the import of Mg 2؉ , could suppress defects in both the formation of the 70S ribosome and growth caused by the absence of L34. Interestingly, the Mg 2؉ content was lower in the ⌬rpmH cells than in the wild type, and the Mg 2؉ content in the ⌬rpmH cells was restored by either the disruption of yhdP or overexpression of mgtE. In vitro experiments on subunit association demonstrated that 50S subunits that lacked L34 could form 70S ribosomes only at a high concentration of Mg 2؉ . These results showed that L34 is required for efficient 70S ribosome formation and that L34 function can be restored partially by Mg 2؉ . In addition, the Mg 2؉ content was consistently lower in mutants that contained significantly reduced amounts of the 70S ribosome, such as the ⌬rplA (L1) and ⌬rplW (L23) strains and mutant strains with a reduced number of copies of the rrn operon. Thus, the results indicated that the cellular Mg 2؉ content is influenced by the amount of 70S ribosomes.T he eubacterial ribosome (70S), which plays a central role in protein synthesis, is composed of a small (30S) subunit and a large (50S) subunit. The small subunit is comprised of the 16S rRNA and more than 20 proteins, whereas the large subunit is comprised of the 23S and 5S rRNAs and more than 30 proteins (1, 2). The molecular mechanisms of translation have been elucidated in detail by the convergence of various approaches, including crystal structure analysis (3-8). The individual functions of several ribosomal proteins have also been elucidated by biochemical and genetic analyses, including reconstitution and mutational analysis. For example, ribosomal protein L2 plays important roles in the assembly of the ribosomal subunits, binding of the tRNA to the A and P sites, peptidyltransferase activity, and formation of the peptide bond (9-13). However, in general, disruption of the genes that encode the ribosomal proteins has been avoided as a means of identifying protein function, because these genes, which are highly conserved in bacteria, have been considered essential for cell proliferation (14). Nonetheless, recently, it was found that 22 of the 54 Escherichia coli genes for ribosomal proteins could be deleted on an individual basis (15,16). We have also shown that out of the 57 ribosomal-protein-encoding genes that have been annotated in the Gram-positive bacterium Bacillus subtilis, at least 22 genes are not individually essential for cell proliferation (17). The rpmH gene encoding ribosomal protein L34, which is a component of the large subunit, is one of the...

Mg 2+ , K + , and the Ribosome

“…Bound and free Mg 2ϩ are in equilibrium, and it is conceivable that a change of the Mg 2ϩ fraction bound to ribosomes will affect the free Mg 2ϩ concentration. A surprising link between intracellular Mg 2ϩ concentration and ribosome amount is reported by Akanuma et al (32) in this issue of the Journal of Bacteriology. They analyzed a mutant of Bacillus subtilis in which the gene for the ribosomal protein L34 was deleted, one of 22 genes of ribosomal proteins which could be knocked out without losing viability (33).…”

“…Akanuma et al (32) determined the total Mg 2ϩ amount with the help of a dye which forms colored chelates with Mg 2ϩ . A 100 mM concentration was observed in wild-type cells (very similar to the total Mg 2ϩ content of an E. coli cell [30]); more than 3 times less Mg 2ϩ was found in the L34-lacking mutant, which could be partially healed when, in addition, either YdpH was deleted or MgtE was overexpressed.…”

Altering the Divalent Metal Ion Preference of RNase E

“…The physiological ion preference of RNase E. As noted in the Introduction, the intracellular concentration of Mg 2ϩ in E. coli can exceed 100 mM but almost all (ϳ98%) is chelated by a variety of macromolecules or small molecules (e.g., organic acids, nucleotides, and phospholipids [8][9][10][11][12]). This implies that the activity of RNase E may normally be limited by the availability of divalent metal ions.…”

“…The intracellular concentration of Mg 2ϩ in E. coli can reach almost 200 mM (8, 9); however, most Mg 2ϩ ions are chelated (e.g., by ribosomes [10,11]) and the free concentration is only 1 to 5 mM (8)(9)(10)12). This implies that the activity of RNase E may be limited by the availability of divalent metal ions.…”

When Too Much ATP Is Bad for Protein Synthesis

“…Assuming a cell volume of 1 μm 3 [25], the entire pool of 70,000 ribosomes chelates at least 12 mM Mg 2+ , the equivalent of 25% of the total Mg 2+ in the cell (Table 1 and Chart 1). Indeed, experiments carried out in Bacillus subtilis indicate that there is a direct correlation between Mg 2+ content and number of ribosomes [26]. Given that total Mg 2+ levels vary from 30 mM in mammalian tissues [8] to 75 -100 mM in bacteria [26,27] (of which 50 -65 mM are intracellular, and 25 -35 mM, or one third, are bound to the components of the cell wall [28]), the translation apparatus traps a significant fraction of the intracellular Mg 2+ .…”