Proteomic study of the Bacillus subtilis ribosome: Finding of zinc-dependent replacement for ribosomal protein L31 paralogues

Abstract

The ribosome is a universal molecular machinery of translation comprising three kinds of ribosomal RNA (rRNA) and many ribosomal proteins (r-proteins). The functional domains of the ribosome, which are involved in the decoding of genetic information in messenger RNA and formation of peptide bonds, consist entirely of rRNAs (reviewed in Ramakrishnan, 2002). As rRNAs are subjected to strong selective pressure and are highly conserved, they have been used as a reference molecule in phylogeny. In contrast, r-proteins show more diversity in their sequences and composition, compared to rRNAs.Recent development of genomics and proteomics has enabled an examination of r-protein genes in prokaryotic, eukaryotic and organelle genomes. A recent comprehensive study of ribosomal genes in completed genomes revealed 57 r-protein families in the bacterial domain, 68 in the archaeal domain and 78 in the eukaryotic domain (Lecompte et al., 2002 Recent advanced studies of genomics and proteomics have revealed the variation and diversity of ribosomal proteins (r-proteins) in different organisms and organelles. Radical free and highly reducing (RFHR) two-dimensional (2-D) electrophoresis is known to be powerful for separating ribosomal proteins that are usually small and basic, and not separated well by standard 2-D electrophoresis. Using the RFHR method, we investigated the protein profile of the Bacillus subtilis ribosomes by a proteomic approach. We found that two L31 paralogue proteins (RpmE and YtiA) showed different temporal expression patterns in the ribosomes. The RpmE protein, which is an L31 variant with a Zn-binding motif, binds one zinc ion at the motif, which is required for stabilization of the protein in the cell. On the other hand, the expression of the ytiA gene, which encodes another L31 variant (YtiA) without the Zn-binding motif, is negatively controlled by the zinc-specific transcriptional repressor Zur and is likely induced by zinc starvation. This article reviews the recent findings that replacement of two types of L31 proteins in the ribosome is controlled by the intracellular zinc concentration.

Towards an Elucidation of the Roles of the Ribosome during Different Growth Phases inBacillus subtilis

“…In addition, newly synthesized RpmE cannot bind zinc, and thus is unstable under these conditions. 60,62,67) Although a similar model was predicted by in silico analysis, 68) we were able to provide the first experimental evidence of actual switching between the two types of L31.…”

“…59) Given that a standard apparatus for this method is available commercially from Nihon-Eido (Tokyo) and is cost effective, we decided to utilize this method for proteomic analysis. 60) In order to identify the constituent proteins of the ribosome of B. subtilis during different growth phases, B. subtilis cells were incubated in CSM, a semisynthetic competence and sporulation medium, and then harvested at various stages of growth. During growth in CSM, competence was initiated just after the cells became unable to grow exponentially, and then sporulation initiated after the competence phase.…”

“…2). 60,62,67) During exponential growth in CSM, which is considered to constitute zinc-sufficient conditions, RpmE contains a zinc ion bound to the CxxC motif and thus is stable. In addition, active Zur protein binds to a motif in the promoter of ytiA, the so-called ''Zur box,'' which is highly conserved among the genes regulated by Zur, 65) and thus represses the expression of ytiA.…”

Characterization of Zn(II)-responsive ribosomal proteins YkgM and L31 in E. coli

“…Using results on B. subtilis as an analogy, the C− YkgM is produced in Zn(II) starvation conditions and replaces the C+ L31 on the ribosome, thereby releasing the Zn(II) bound to L31 [20, 38, 39]. To evaluate whether L31 is in fact a Zn(II)-binding protein, the L31 gene was ligated into pIAD14 [24], which resulted in the production of a MBP-L31 fusion protein.…”

Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins

“…Many of the proteomic strategies used to study the ribosome have included various forms of gel electrophoresis with MS (13)(14)(15). Although some great successes have been had using these methods, for many ribosomal studies the demand is for automated methods with a higher degree of reproducibility.…”