Authentic precursors to ribosomal subunits accumulate in Escherichia coli in the absence of functional DnaK chaperone

Escherichia coli dnaK-ts mutants are defective in the late stages of ribosome biogenesis at high temperature. Here, we show that the 21S, 32S and 45S ribosomal particles that accumulate in the dnaK756-ts mutant at 44 degrees C contain unprocessed forms of their 16S and 23S rRNAs (partially processed in the case of 45S particles). Their 5S rRNA stoichiometry and ribosomal protein composition are typical of the genuine ribosomal precursors found in a wild-type (dnaK+) strain. Despite the lack of a functional DnaK, a very slow maturation of these 21S, 32S and 45S particles to structurally and functionally normal 30S and 50S ribosomal subunits still occurs at high temperature. This conversion is accompanied by the processing of p16S and p23S rRNAs to their mature forms. We conclude that: (i) 21S, 32S and 45S particles are not dead-end particles, but true precursors to active ribosomes (21S particles are converted to 30S subunits, and 32S and 45S to 50S subunits); (ii) DnaK is not absolutely necessary for ribosome biogenesis, but accelerates the late steps of this process considerably at high temperature; and (iii) 23S rRNA processing depends on the stage reached in the stepwise assembly of the 50S subunit, not directly on DnaK.     

The 60S associated ribosome biogenesis factor LSG1‐2 is required for 40S maturation in Arabidopsis thaliana

Ribosome biogenesis involves a large ensemble of trans‐acting factors, which catalyse rRNA processing, ribosomal protein association and ribosomal subunit assembly. The circularly permuted GTPase Lsg1 is such a ribosome biogenesis factor, which is involved in maturation of the pre‐60S ribosomal subunit in yeast. We identified two orthologues of Lsg1 in Arabidopsis thaliana. Both proteins differ in their C‐terminus, which is highly charged in atLSG1‐2 but missing in atLSG1‐1. This C‐terminus of atLSG1‐2 contains a functional nuclear localization signal in a part of the protein that also targets atLSG1‐2 to the nucleolus. Furthermore, only atLSG1‐2 is physically associated with ribosomes suggesting its function in ribosome biogenesis. Homozygous T‐DNA insertion lines are viable for both LSG1 orthologues. In plants lacking atLSG1‐2 18S rRNA precursors accumulate and a 20S pre‐rRNA is detected, while the amount of pre‐rRNAs that lead to the 25S and 5.8S rRNA is not changed. Thus, our results suggest that pre‐60S subunit maturation is important for the final steps of pre‐40S maturation in plants. In addition, the lsg1‐2 mutants show severe developmental defects, including triple cotyledons and upward curled leaves, which link ribosome biogenesis to early plant and leaf development.     

Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine

The late stages of 30S and 50S ribosomal subunits biogenesis have been studied in a wild-type (wt) strain of Escherichia coli (MC4100) subjected to a severe heat stress (45-46°C). The 32S and 45S ribosomal particles (precursors to 50S subunits) and 21S ribosomal particles (precursors to 30S subunits) accumulate under these conditions. They are authentic precursors, not degraded or dead-end particles. The 21S particles are shown, by way of a modified 3'5' RACE procedure, to contain 16S rRNA unprocessed, or processed at its 5' end, and not at the 3' end. This implies that maturation of 16S rRNA is ordered and starts at its 5'-terminus, and that the 3'-terminus is trimmed at a later step. This observation is not limited to heat stress conditions, but it also can be verified in bacteria growing at a normal temperature (30°C), supporting the idea that this is the general pathway. Assembly defects at very high temperature are partially compensated by plasmid-driven overexpression of the DnaK/DnaJ chaperones. The ribosome assembly pattern in wt bacteria under a severe heat stress is therefore reminiscent of that observed at lower temperatures in E. coli mutants lacking the chaperones DnaK or DnaJ.     

The RNA‐binding protein Hfq is important for ribosome biogenesis and affects translation fidelity

Ribosome biogenesis is a complex process involving multiple factors. Here, we show that the widely conserved RNA chaperone Hfq, which can regulate sRNA‐mRNA basepairing, plays a critical role in rRNA processing and ribosome assembly in Escherichia coli. Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA. Hfq assists ribosome assembly and associates with pre‐30S particles but not with mature 30S subunits. Inactivation of Hfq strikingly decreases the pool of mature 70S ribosomes. The reduction in ribosome levels depends on residues located in the distal face of Hfq but not on residues found in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that Hfq‐mediated regulation of ribosomes is independent of its function as sRNA‐regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of the Sm‐like protein Hfq beyond its function in small RNA‐mediated regulation and unveils a novel role of Hfq as crucial in ribosome biogenesis and translation.     

The function of RH22, a DEAD RNA helicase, in the biogenesis of the 50S ribosomal subunits of Arabidopsis chloroplasts

The chloroplast ribosome is a large and dynamic ribonucleoprotein machine that is composed of the 30S and 50S subunits. Although the components of the chloroplast ribosome have been identified in the last decade, the molecular mechanisms driving chloroplast ribosome biogenesis remain largely elusive. Here, we show that RNA helicase 22 (RH22), a putative DEAD RNA helicase, is involved in chloroplast ribosome assembly in Arabidopsis (Arabidopsis thaliana). A loss of RH22 was lethal, whereas a knockdown of RH22 expression resulted in virescent seedlings with clear defects in chloroplast ribosomal RNA (rRNA) accumulation. The precursors of 23S and 4.5S, but not 16S, rRNA accumulated in rh22 mutants. Further analysis showed that RH22 was associated with the precursors of 50S ribosomal subunits. These results suggest that RH22 may function in the assembly of 50S ribosomal subunits in chloroplasts. In addition, RH22 interacted with the 50S ribosomal protein RPL24 through yeast two-hybrid and pull-down assays, and it was also bound to a small 23S rRNA fragment encompassing RPL24-binding sites. This action of RH22 may be similar to, but distinct from, that of SrmB, a DEAD RNA helicase that is involved in the ribosomal assembly in Escherichia coli, which suggests that DEAD RNA helicases and rRNA structures may have coevolved with respect to ribosomal assembly and function.     

Ribosome assembly in Escherichia coli strains lacking the RNA helicase DeaD/CsdA or DbpA

Ribosome subunit assembly in bacteria is a fast and efficient process. Among the nonribosomal proteins involved in ribosome biogenesis are RNA helicases. We describe ribosome biogenesis in Escherichia coli strains lacking RNA helicase DeaD (CsdA) or DbpA. Ribosome large subunit assembly intermediate particles (40S) accumulate at 25 degrees C and at 37 degrees C in the absence of DeaD but not without DbpA. 23S rRNA is incompletely processed in the 40S and 50S particles of the DeaD(-) strain. Pulse labeling showed that the 40S particles are converted nearly completely into functional ribosomes. The rate of large ribosomal subunit assembly was reduced about four times in DeaD-deficient cells. Functional activity tests of the ribosomal particles demonstrated that the final step of 50S assembly, the activation step, was affected when DeaD was not present. The results are compatible with the model that predicts multiple DeaD-catalyzed structural transitions of the ribosome large subunit assembly.     

Era and RbfA have overlapping function in ribosome biogenesis in Escherichia coli

A cold-shock protein, RbfA (ribosome-binding factor A), is essential for cell growth at low temperature. In an rbfA-deletion strain, 30S and 50S ribosomal subunits increase relative to 70S monosomes with concomitant accumulation of a precursor 16S rRNA (17S rRNA). Recently, we have reported that overexpression of Era, an essential GTP-binding protein, suppresses not only the cold-sensitive cell growth but also defective ribosome biogenesis in the rbfA-deletion strain. Here, in order to elucidate how RbfA and Era functionally overlap, we characterized a cold-sensitive Era mutant (a point mutation at the Glu-200 to Lys; E200K) which shows a similar phenotype as the rbfA-deletion strain; accumulation of free ribosome subunits and 17S rRNA. To examine the effect of E200K in the rbfA-deletion strain, we constructed an E200K-inducible expression system. Interestingly, unlike wild-type Era, overexpression of Era(E200K) protein in the rbfA-deletion strain severely inhibited cell growth even at permissive temperature with further concomitant reduction of 16S rRNA. Purified Era(E200K) protein binds to 30S ribosomal subunits in a nucleotide-dependent manner like wild-type Era and retains both GTPase and autophosphorylation activities. Furthermore, we isolated spontaneous revertants of the E200K mutant. These revertants partially suppressed the accumulation of 17S rRNA. All the spontaneous mutations were found to result in higher Era(E200K) expression. These results suggest that the Era(E200K) protein has an impaired function in ribosome biogenesis without losing its ribosome binding activity. The severe growth defect caused by E200K in the rbfA-deletion strain may be due to competition between intrinsic wild-type Era and overexpressed Era(E200K) for binding to 30S ribosomal subunits. We propose that Era and RbfA have an overlapping function that is essential for ribosome biogenesis, and that RbfA becomes dispensable only at high temperatures because Era can complement its function only at higher temperatures.     

Analysis of ribosomal inter‐subunit sites as targets for complementary oligonucleotides

The bacterial ribosome has many functional ribosomal RNA (rRNA) sites. We have computationally analyzed the rRNA regions involved in the interactions between the 30S and 50S subunits. Various properties of rRNA such as solvent accessibility, opening energy, hydrogen bonding pattern, van der Waals energy, thermodynamic stability were determined. Based on these properties we selected rRNA targets for hybridization with complementary 2′‐O‐methyl oligoribonucleotides (2′‐OMe RNAs). Further, the inhibition efficiencies of the designed ribosome‐interfering 2′‐OMe RNAs were tested using a β‐galactosidase assay in a translation system based on the E. coli extract. Several of the oligonucleotides displayed IC₅₀ values below 1 μM, which were in a similar range as those determined for known ribosome inhibitors, tetracycline and pactamycin. The calculated opening and van der Waals stacking energies of the rRNA targets correlated best with the inhibitory efficiencies of 2′‐OMe RNAs. Moreover, the binding affinities of several oligonucleotides to both 70S ribosomes and isolated 30S and 50S subunits were measured using a double‐filter retention assay. Further, we applied heat‐shock chemical transformation to introduce 2′‐OMe RNAs to E. coli cells and verify inhibition of bacterial growth. We observed high correlation between IC₅₀ in the cell‐free extract and bacterial growth inhibition. Overall, the results suggest that the computational analysis of potential rRNA targets within the conformationally dynamic regions of inter‐subunit bridges can help design efficient antisense oligomers to probe the ribosome function.     

Interaction network of the ribosome assembly machinery from a eukaryotic thermophile

Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre‐ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre‐ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (～180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in‐depth analysis of their protein–protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2‐hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein–protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre‐ribosome factors forming the ctUTP‐A and ctUTP‐B modules, and the Brix‐domain containing assembly factors of the pre‐60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.     

Mutations of ribosomal protein S5 suppress a defect in late-30S ribosomal subunit biogenesis caused by lack of the RbfA biogenesis factor

The in vivo assembly of ribosomal subunits requires assistance by maturation proteins that are not part of mature ribosomes. One such protein, RbfA, associates with the 30S ribosomal subunits. Loss of RbfA causes cold sensitivity and defects of the 30S subunit biogenesis and its overexpression partially suppresses the dominant cold sensitivity caused by a C23U mutation in the central pseudoknot of 16S rRNA, a structure essential for ribosome function. We have isolated suppressor mutations that restore partially the growth of an RbfA-lacking strain. Most of the strongest suppressor mutations alter one out of three distinct positions in the carboxy-terminal domain of ribosomal protein S5 (S5) in direct contact with helix 1 and helix 2 of the central pseudoknot. Their effect is to increase the translational capacity of the RbfA-lacking strain as evidenced by an increase in polysomes in the suppressed strains. Overexpression of RimP, a protein factor that along with RbfA regulates formation of the ribosome''s central pseudoknot, was lethal to the RbfA-lacking strain but not to a wild-type strain and this lethality was suppressed by the alterations in S5. The S5 mutants alter translational fidelity but these changes do not explain consistently their effect on the RbfA-lacking strain. Our genetic results support a role for the region of S5 modified in the suppressors in the formation of the central pseudoknot in 16S rRNA.     

Nuclear export and cytoplasmic maturation of ribosomal subunits

Based on the characterization of ribosome precursor particles and associated trans-acting factors, a biogenesis pathway for the 40S and 60S subunits has emerged. After nuclear synthesis and assembly steps, pre-ribosomal subunits are exported through the nuclear pore complex in a Crm1- and RanGTP-dependent manner. Subsequent cytoplasmic biogenesis steps of pre-60S particles include the facilitated release of several non-ribosomal proteins, yielding fully functional 60S subunits. Cytoplasmic maturation of 40S subunit precursors includes rRNA dimethylation and pre-rRNA cleavage, allowing 40S subunits to achieve translation competence. We review current knowledge of nuclear export and cytoplasmic maturation of ribosomal subunits.     

The multifunctional nucleolus

The nucleolus is a distinct subnuclear compartment that was first observed more than 200 years ago. Nucleoli assemble around the tandemly repeated ribosomal DNA gene clusters and 28S, 18S and 5.8S ribosomal RNAs (rRNAs) are transcribed as a single precursor, which is processed and assembled with the 5S rRNA into ribosome subunits. Although the nucleolus is primarily associated with ribosome biogenesis, several lines of evidence now show that it has additional functions. Some of these functions, such as regulation of mitosis, cell-cycle progression and proliferation, many forms of stress response and biogenesis of multiple ribonucleoprotein particles, will be discussed, as will the relation of the nucleolus to human diseases.     

Synthetic Lethality in the Tobacco Plastid Ribosome and Its Rescue at Elevated Growth Temperatures

Consistent with their origin from cyanobacteria, plastids (chloroplasts) perform protein biosynthesis on bacterial-type 70S ribosomes. The plastid genomes of seed plants contain a conserved set of ribosomal protein genes. Three of these have proven to be nonessential for translation and, thus, for cellular viability: rps15, rpl33, and rpl36. To help define the minimum ribosome, here, we examined whether more than one of these nonessential plastid ribosomal proteins can be removed from the 70S ribosome. To that end, we constructed all possible double knockouts for the S15, L33, and L36 ribosomal proteins by stable transformation of the tobacco (Nicotiana tabacum) plastid genome. We find that, although S15 and L33 function in different ribosomal particles (30S and 50S, respectively), their combined deletion from the plastid genome results in synthetic lethality under autotrophic conditions. Interestingly, the lethality can be overcome by growth under elevated temperatures due to an improved efficiency of plastid ribosome biogenesis. Our results reveal functional interactions between protein and RNA components of the 70S ribosome and uncover the interdependence of the biogenesis of the two ribosomal subunits. In addition, our findings suggest that defining a minimal set of plastid genes may prove more complex than generally believed.     

Ribosomal proteins: XLIII. In vivo assembly of Escherichia coli ribosomal proteins

Isolation of ribosomal precursors from Escherichia coli K12 is described. The RNA and protein content of the precursor particles was determined.One physiologically stable precursor was found for the 30 S subunit. The assembly scheme is as follows: p16 S RNA + 9 proteins → p30 S (“21 S” precursor) p30 S + 12 proteins → 30 S subunit where p is precursor.Each of the two precursors for the 50 S subunit, P₁50 S and p₂50 S (“32 S” and “43 S” precursors, respectively), contains p5 S + p23 S RNA's in a 1:1 molar ratio. The assembly scheme is as follows: p23 S RNA + p5 S RNA + 16 or 17 proteins → p₁50 S

In contrast to the p₂50 S precursor the p₁50 S precursor is not similar to any core particles, which were obtained by treating 50 S subunits with different concentrations of LiCl or CsCl.The precursors p30 S and p₂50 S can be converted into active 30 S and 50 S sub-units, respectively, by incubation at 42 °C in the presence of ribosomal proteins and under RNA methylating conditions.     

Roles of elusive translational GTPases come to light and inform on the process of ribosome biogenesis in bacteria

Protein synthesis relies on several translational GTPases (trGTPases), related proteins that couple the hydrolysis of GTP to specific molecular events on the ribosome. Most bacterial trGTPases, including IF2, EF‐Tu, EF‐G and RF3, play well‐known roles in translation. The cellular functions of LepA (also termed EF4) and BipA (also termed TypA), conversely, have remained enigmatic. Recent studies provide compelling in vivo evidence that LepA and BipA function in biogenesis of the 30S and 50S subunit respectively. These findings have important implications for ribosome biogenesis in bacteria. Because the GTPase activity of each of these proteins depends on interactions with both ribosomal subunits, some portion of 30S and 50S assembly must occur in the context of the 70S ribosome. In this review, we introduce the trGTPases of bacteria, describe the new functional data on LepA and BipA, and discuss the how these findings shape our current view of ribosome biogenesis in bacteria.     

Defective processing of ribosomal precursor RNA in Saccharomyces cerevisiae.

Saccharomyces cerevisiae (strain A224A) has an abnormal distribution of cytoplasmic ribosomal subunits when grown at 36 degrees C, with sucrose-gradient analysis of extracts revealing an apparent excess of material sedimenting at 60 S. This abnormality is not observed at either 23 degrees C or 30 degrees C. At 36 degrees C the defect(s) is expressed as a slowed conversion of 20 S ribosomal precursor RNA to mature 18 S rRNA, although the corresponding maturation of 27 S ribosomal precursor RNA to mature 25 S rRNA is normal. Studies on this yeast strain and on mutants derived from it may help to elucidate the role(s) of individual ribosomal components in controlling ribosome biogenesis in eukaryotes.