The hibernation promoting factor of Betaproteobacteria Comamonas testosteroni cannot induce 100S ribosome formation but stabilizes 70S ribosomal particles.

Bacteria use several means to survive under stress conditions such as nutrient depletion. One such response is the formation of hibernating 100S ribosomes, which are translationally inactive 70S dimers. In Gammaproteobacteria (Enterobacterales), 100S ribosome formation requires ribosome modulation factor (RMF) and short hibernation promoting factor (HPF), whereas it is mediated by only long HPF in the majority of bacteria. Here, we investigated the role of HPFs of Comamonas testosteroni, which belongs to the Betaproteobacteria with common ancestor to the Gammaproteobacteria. C. testosteroni has two genes of HPF homologs of differing length (CtHPF-125 and CtHPF-119). CtHPF-125 was induced in the stationary phase, whereas CtHPF-119 conserved in many other Betaproteobacteria was not expressed in the culture conditions used here. Unlike short HPF and RMF, and long HPF, CtHPF-125 could not form 100S ribosome. We first constructed the deletion mutant of Cthpf-125 gene. When the deletion mutant grows in the stationary phase, 70S particles were degraded faster than in the wild strain. CtHPF-125 contributes to stabilizing the 70S ribosome. CtHPF-125 and CtHPF-119 both inhibited protein synthesis by transcription-translation in vitro. Our findings suggest that CtHPF-125 binds to ribosome, and stabilizes 70S ribosomes, inhibits translation without forming 100S ribosomes and supports prolonging life.

The Discovery of Ribosomal Protein bL31 from Escherichia coli: A Long Story Revisited.

Ribosomal protein bL31 in Escherichia coli was initially detected as a short form (62 amino acids) using Kaltschmidt and Wittmann's two-dimensional polyacrylamide gel electrophoresis (2D PAGE), but the intact form (70 amino acids) was subsequently identified by means of Wada's improved radical-free and highly reducing (RFHR) 2D PAGE, which was consistent with the analysis of its encoding gene rpmE. Ribosomes routinely prepared from the K12 wild-type strain contained both forms of bL31. ΔompT cells, which lack protease 7, only contained intact bL31, suggesting that protease 7 cleaves intact bL31 and generates short bL31 during ribosome preparation from wild-type cells. Intact bL31 was required for subunit association, and its eight cleaved C-terminal amino acids contributed to this function. 70S ribosomes protected bL31 from cleavage by protease 7, but free 50S did not. In vitro translation was assayed using three systems. The translational activities of wild-type and ΔrpmE ribosomes were 20% and 40% lower than those of ΔompT ribosomes, which contained one copy of intact bL31. The deletion of bL31 reduces cell growth. A structural analysis predicted that bL31 spans the 30S and 50S subunits, consistent with its functions in 70S association and translation. It is important to re-analyze in vitro translation with ribosomes containing only intact bL31.

Functional Sites of Ribosome Modulation Factor (RMF) Involved in the Formation of 100S Ribosome.

One of the important cellular events in all organisms is protein synthesis, which is catalyzed by ribosomes. The ribosomal activity is dependent on the environmental situation of the cell. Bacteria form 100S ribosomes, lacking translational activity, to survive under stress conditions such as nutrient starvation. The 100S ribosome is a dimer of two 70S ribosomes bridged through the 30S subunits. In some pathogens of gammaproteobacteria, such as Escherichia coli, Yersinia pestis, and Vibrio cholerae, the key factor for ribosomal dimerization is the small protein, ribosome modulation factor (RMF). When ribosomal dimerization by RMF is impaired, long-term bacterial survival is abolished. This shows that the interconversion system between active 70S ribosomes and inactive 100S ribosomes is an important survival strategy for bacteria. According to the results of several structural analyses, RMF does not directly connect two ribosomes, but binds to them and changes the conformation of their 30S subunits, thus promoting ribosomal dimerization. In this study, conserved RMF amino acids among 50 bacteria were selectively altered by mutagenesis to identify the residues involved in ribosome binding and dimerization. The activities of mutant RMF for ribosome binding and ribosome dimerization were measured using the sucrose density gradient centrifugation (SDGC) and western blotting methods. As a result, some essential amino acids of RMF for the ribosomal binding and dimerization were elucidated. Since the induction of RMF expression inhibits bacterial growth, the data on this protein could serve as information for the development of antibiotic or bacteriostatic agents.

YkgM and YkgO maintain translation by replacing their paralogs, zinc-binding ribosomal proteins L31 and L36, with identical activities.

When a cell is zinc-deficient, ykgM and ykgO, which encode paralogs of the zinc-binding ribosomal proteins L31 and L36, are expressed from the ykgM operon, which is ordinarily held inactive by the Zur repressor. In ribosomes lacking L31, ribosomal subunit association is weakened, resulting in reduced in vitro translation and the deletion mutants of rpmE, the gene encoding L31, forming small colonies. We isolated four suppressor mutants of ∆rpmE that formed normal colonies. All four mutation sites were located in zur, and ribosomes of zur mutant cells contained one copy of YkgM and had translational activities equivalent to those of ribosomes containing L31. L36 is highly conserved among bacteria, chloroplast and mitochondria. Analysis of a deletion mutant of rpmJ, which encodes L36, suggested that L36 is involved in late assembly of the 50S particle, in vitro translation and cell growth. In zur mutant cells lacking rpmJ, the paralog YkgO was expressed and took over the functions of L36. zur mutant cells contained four types of ribosomes containing combinations of L31 or YkgM, and L36 or YkgO. Copy numbers of L31 and YkgM, and L36 and YkgO, summed to 1, indicating that each paralog pair shares a binding site.

Ribosomal protein L31 in Escherichia coli contributes to ribosome subunit association and translation, whereas short L31 cleaved by protease 7 reduces both activities.

Ribosomes routinely prepared from Escherichia coli strain K12 contain intact (70 amino acids) and short (62 amino acids) forms of ribosomal protein L31. By contrast, ribosomes prepared from ompT mutant cells, which lack protease 7, contain only intact L31, suggesting that L31 is cleaved by protease 7 during ribosome preparation. We compared ribosomal subunit association in wild-type and ompT - strains. In sucrose density gradient centrifugation under low Mg2+ , 70S content was very high in ompT - ribosomes, but decreased in the wild-type ribosomes containing short L31. In addition, ribosomes lacking L31 failed to associate ribosomal subunits in low Mg2+ . Therefore, intact L31 is required for subunit association, and the eight C-terminal amino acids contribute to the association function. In vitro translation was assayed using three different systems. Translational activities of ribosomes lacking L31 were 40% lower than those of ompT - ribosomes with one copy of intact L31, indicating that L31 is involved in translation. Moreover, in the stationary phase, L31 was necessary for 100S formation. The strain lacking L31 grew very slowly. A structural analysis predicted that the L31 protein spans the 30S and 50S subunits, consistent with the functions of L31 in 70S association, 100S formation, and translation.

Conservation of two distinct types of 100S ribosome in bacteria.

In bacteria, 70S ribosomes (consisting of 30S and 50S subunits) dimerize to form 100S ribosomes, which were first discovered in Escherichia coli. Ribosome modulation factor (RMF) and hibernation promoting factor (HPF) mediate this dimerization in stationary phase. The 100S ribosome is translationally inactive, but it dissociates into two translationally active 70S ribosomes after transfer from starvation to fresh medium. Therefore, the 100S ribosome is called the 'hibernating ribosome'. The gene encoding RMF is found widely throughout the Gammaproteobacteria class, but is not present in any other bacteria. In this study, 100S ribosome formation in six species of Gammaproteobacteria and eight species belonging to other bacterial classes was compared. There were several marked differences between the two groups: (i) Formation of 100S ribosomes was mediated by RMF and short HPF in Gammaproteobacteria species, similar to E. coli, whereas it was mediated only by long HPF in the other bacterial species; (ii) RMF/short HPF-mediated 100S ribosome formation occurred specifically in stationary phase, whereas long HPF-mediated 100S ribosome formation occurred in all growth phases; and (iii) 100S ribosomes formed by long HPF were much more stable than those formed by RMF and short HPF.

YqjD is an inner membrane protein associated with stationary-phase ribosomes in Escherichia coli.

Here, we provide evidence that YqjD, a hypothetical protein of Escherichia coli, is an inner membrane and ribosome binding protein. This protein is expressed during the stationary growth phase, and expression is regulated by stress response sigma factor RpoS. YqjD possesses a transmembrane motif in the C-terminal region and associates with 70S and 100S ribosomes at the N-terminal region. Interestingly, E. coli possesses two paralogous proteins of YqjD, ElaB and YgaM, which are expressed and bind to ribosomes in a similar manner to YqjD. Overexpression of YqjD leads to inhibition of cell growth. It has been suggested that YqjD loses ribosomal activity and localizes ribosomes to the membrane during the stationary phase.

Comparative proteomic analysis of the ribosomes in 5-fluorouracil resistance of a human colon cancer cell line using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis.

Many auxiliary functions of ribosomal proteins (r-proteins) have received considerable attention in recent years. However, human r-proteins have hardly been examined by proteomic analysis. In this study, we isolated ribosomal particles and subsequently compared the proteome of r-proteins between the DLD-1 human colon cancer cell line and its 5-fluorouracil (5-FU)-resistant sub-line, DLD-1/5-FU, using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis, which has a superior ability to separate basic proteins, and we discuss the role of r-proteins in 5-FU resistance. Densitometric analysis was performed to quantify modulated proteins, and protein spots showing significant changes were identified by employing matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Three basic proteins (L15, L37 and prohibitin) which were significantly modulated between DLD-1 and DLD-1/5-FU were identified. Two proteins, L15 and L37, showed down-regulated expression in DLD-1/5-FU in comparison to DLD-1. Prohibitin, which is not an r-protein and is known to be localized in the mitochondria, showed up-regulated expression in DLD-1/5-FU. These 3 proteins may be related to 5-FU resistance.

The involvement of a PPR protein of the P subfamily in partial RNA editing of an Arabidopsis mitochondrial transcript.

C-to-U RNA editing (i.e., alteration of a C in the genomic sequence to U in the transcript) has been confirmed widely in angiosperm organellar genomes. During the C-to-U RNA editing event, incomplete edited transcripts have been observed at many sites in the steady-state mRNA population (partial editing). Here, by using coexpression analysis and the surveillance of whole editing status on the mitochondrial genome, we have revealed that a pentatricopeptide repeat (PPR) protein classified into the P subfamily (PPR596) has site-specific influence on the efficiency of C-to-U RNA editing events at partial editing sites on the Arabidopsis thaliana mitochondrial genome. Previous works have revealed that PPR proteins classified into the PLS subfamily containing the E or E and DYW motif are involved in RNA editing as trans-factors; they are believed to recruit deaminase at editing sites. In contrast with the mutant analyses of PLS-subfamily PPR proteins, the editing efficiency at rps3eU1344SS was revealed to be significantly increased in ppr596 mutants. Our study implies P-subfamily PPR protein is involved in the control of the degree of partial editing.

Formation of 100S ribosomes in Staphylococcus aureus by the hibernation promoting factor homolog SaHPF.

In the stationary growth phase of Escherichia coli, the 70S ribosomes are dimerized by the ribosome modulation factor (RMF) and hibernation promoting factor (HPF) proteins to form 100S ribosomes, which lose translational activity. In this study we found 100S ribosomes in the gram-positive bacterium Staphylococcus aureus, which has an HPF homolog (named SaHPF) but no RMF homolog. Unlike in E. coli, 100S ribosomes exist in all growth phases of S. aureus, with the highest levels at the transition from the exponential phase to the stationary phase. To find the key factors involved in 100S formation, we analyzed proteins associated with crude ribosomes using radical-free and highly reducing 2-D PAGE and MALDI TOF/MS. Only the SaHPF levels changed in parallel with the changes in 100S levels. SaHPF bound preferentially to 70S components in 100S ribosomes, with a molar ratio of 1 : 1 relative to the 70S, but some SaHPF was also detected in free 70S ribosomes. High-salt washing of the crude ribosomes released SaHPF and dissociated the 100S ribosomes to their 70S components. When these 70S components were incubated with purified SaHPF in vitro, they re-associated to form 100S. These results suggest that SaHPF is a key protein involved in 100S ribosome formation in S. aureus.

Solution structure of the E. coli ribosome hibernation promoting factor HPF: Implications for the relationship between structure and function.

The 70S Escherichia coli ribosome dimerizes to form an inactive 100S ribosome during stationary phase, which is called "ribosome hibernation". The hibernation promoting factor HPF plays a crucial role in 100S ribosome formation. However, YfiA, a known paralog of HPF inhibits 100S formation, although it shares high sequence similarity. Here, we report the first solution structure of HPF as determined by multi-dimensional NMR. HPF adopts betaalphabetabetabetaalpha-fold and the overall structure is similar to YfiA as expected. However, detailed structure comparison based on the determined structure in this study revealed that there are remarkable differences around the C-terminal portion of helix alpha2, which is not predicted by homology modeling. Furthermore, some acidic residues conserved only in HPF are located at the rim of the common basic patch.

Activities of Escherichia coli ribosomes in IF3 and RMF change to prepare 100S ribosome formation on entering the stationary growth phase.

The canonical ribosome cycle in bacteria consists of initiation, elongation, termination, and recycling stages. After the recycling stage, initiation factor 3 (IF3) stabilizes ribosomal dissociation by binding to 30S subunits for the next round of translation. On the other hand, during the stationary growth phase, it has been elucidated that Escherichia coli ribosomes are dimerized (100S ribosome formation) by binding ribosome modulation factor (RMF) and hibernation promoting factor (HPF), leading to a hibernation stage. This indicates that 100S ribosomes are formed after these factors are scrambled for ribosomes concomitantly with transition from the log phase to the stationary phase. In this study, to elucidate the ribosomal events before 100S ribosome formation, the relationships between protein factors (RMF and HPF) involved in 100S ribosome formation and IF3 involved in initiation complex formation were examined. As a result of in vitro assays, it was found that ribosomal dissociation activity by IF3 fell, and that ribosomal dimerization activity by RMF and HPF was elevated more when using stationary-phase ribosomes than when using log-phase ribosomes. This suggests that ribosomes change into forms which are hard to bind with IF3 and easy to form 100S ribosomes by RMF and HPF concomitantly with transition from the log phase to the stationary phase.

Role of HPF (hibernation promoting factor) in translational activity in Escherichia coli.

During the stationary phase of growth in Escherichia coli, ribosome modulation factor (RMF) and hibernation promoting factor (HPF) dimerize most 70S ribosomes to form 100S ribosomes. The process of 100S formation has been termed 'ribosomal hibernation'. Here, the contributions of HPF to 100S formation and translation were analysed in vitro. HPF bound to, but did not dimerize the 70S ribosome. RMF dimerized and formed immature 90S ribosomes. Binding of both HPF and RMF converted 90S ribosomes to mature 100S ribosomes, which is consistent with the in vivo data. The role of HPF in in vitro translation also was investigated. In an artificial mRNA poly (U)-dependent phenylalanine incorporation assay, HPF bound to ribosomal particles and inhibited translation. In contrast, in a natural MS2 mRNA-dependent leucine incorporation assay, bound HPF was removed and hardly inhibited normal translation. Multiple alignment and phylogenetic analyses indicates that the hibernation system mediated by the HPF homologue, RMF and 100S ribosome formation may be specific to the proteobacteria gamma group. In contrast, most bacteria have at least one HPF homologue, and these homologues can be classified into three types, long HPF, short HPF and YfiA.

Ribosome binding proteins YhbH and YfiA have opposite functions during 100S formation in the stationary phase of Escherichia coli.

During the stationary phase of Escherichia coli growth, ribosomal structure changes drastically. Proteins RMF, YhbH, YfiA and SRA are expressed and bind to ribosome particles. In a process named 'ribosomal hibernation,' RMF binding induces the dimerization and subsequent inactivation of 70S ribosomes. Here, we examined the functions of YhbH and YfiA in the formation of 70S dimers using deletion mutants of YhbH and YfiA. The yfiA deletion mutant expressed YhbH and RMF in the stationary phase and formed a greater number of 100S particles than the wild-type, showing that YhbH promotes and stabilizes 100S formation. In contrast, the yhbH deletion mutant expressed YfiA and RMF and produced no 70S dimers, suggesting that YfiA prevents 70S dimer formation. Thus, YhbH and YfiA have opposite functions in 70S dimer formation. YhbH and YfiA share 40% sequence homology, suggesting that their binding sites overlap and they compete for a region proximal to the P- and A-sites on 30S subunits. In the yhbH and yfiA double deletion mutant, which expresses only RMF, 70S dimers were observed as 90S particles. Since 100S particles were seen in the yfiA deletion mutant containing RMF and YhbH, YhbH probably converts immature 90S ribosomes into mature 100S particles.