Studies of the interaction of Escherichia coli YjeQ with the ribosome in vitro

Escherichia coli YjeQ represents a conserved group of bacteria-specific nucleotide-binding proteins of unknown physiological function that have been shown to be essential to the growth of E. coli and Bacillus subtilis. The protein has previously been characterized as possessing a slow steady-state GTP hydrolysis activity (8 h(-1)) (D. M. Daigle, L. Rossi, A. M. Berghuis, L. Aravind, E. V. Koonin, and E. D. Brown, Biochemistry 41: 11109-11117, 2002). In the work reported here, YjeQ from E. coli was found to copurify with ribosomes from cell extracts. The copy number of the protein per cell was nevertheless low relative to the number of ribosomes (ratio of YjeQ copies to ribosomes, 1:200). In vitro, recombinant YjeQ protein interacted strongly with the 30S ribosomal subunit, and the stringency of that interaction, revealed with salt washes, was highest in the presence of the nonhydrolyzable GTP analog 5'-guanylylimidodiphosphate (GMP-PNP). Likewise, association with the 30S subunit resulted in a 160-fold stimulation of YjeQ GTPase activity, which reached a maximum with stoichiometric amounts of ribosomes. N-terminal truncation variants of YjeQ revealed that the predicted OB-fold region was essential for ribosome binding and GTPase stimulation, and they showed that an N-terminal peptide (amino acids 1 to 20 in YjeQ) was necessary for the GMP-PNP-dependent interaction of YjeQ with the 30S subunit. Taken together, these data indicate that the YjeQ protein participates in a guanine nucleotide-dependent interaction with the ribosome and implicate this conserved, essential GTPase as a novel factor in ribosome function.     

A novel GTPase activated by the small subunit of ribosome

The GTPase activity of Escherichia coli YjeQ, here named RsgA (ribosome small subunit-dependent GTPase A), has been shown to be significantly enhanced by ribosome or its small subunit. The enhancement of GTPase activity was inhibited by several aminoglycosides bound at the A site of the small subunit, but not by a P site-specific antibiotic. RsgA stably bound the small subunit in the presence of GDPNP, but not in the presence of GTP or GDP, to dissociate ribosome into subunits. Disruption of the gene for RsgA from the genome affected the growth of the cells, which predominantly contained the dissociated subunits having only a weak activation activity of RsgA. We also found that 17S RNA, a putative precursor of 16S rRNA, was contained in the small subunit of the ribosome from the RsgA-deletion strain. RsgA is a novel GTPase that might provide a new insight into the function of ribosome.     

RelA functionally suppresses the growth defect caused by a mutation in the G domain of the essential Der protein

A unique bacterial GTPase, Der, containing two tandem GTP-binding domains, is essential for cell growth and plays a crucial role in a large ribosomal subunit in Escherichia coli. The depletion of Der resulted in accumulation of both large and small ribosomal subunits and also affected the stability of large ribosomal subunits. However, its exact cellular function still remains elusive. Previously, we have shown that two G domain mutants, DerN118D and DerN321D, cannot support cell growth at low temperatures, suggesting that both GTP-binding domains are indispensable. In this study, we show that both Der variants are defective in ribosome biogenesis. Genetic screening of an E. coli genomic library was performed to identify the genes which, when expressed from a multicopy plasmid, can restore the growth defect of the DerN321D mutant at restrictive temperatures. Among seven suppressors isolated, four were located at 62.7 min on the E. coli genomic map, and the gene responsible for the suppression of DerN321D was identified as the relA gene which encodes a ribosome-associated (p)ppGpp synthetase. The synthetic activity of RelA was found to be essential for its DerN321D suppressor activity. Overexpression of RelA in a suppressor strain did not affect the expression of DerN321D but suppressed the polysome defects caused by the DerN321D mutant. This is the first demonstration of suppression of impaired function of Der by a functional enzyme. A possible mechanism of the suppression of DerN321D by RelA overproduction is discussed.     

Cryo-electron microscopy structure of the 30S subunit in complex with the YjeQ biogenesis factor

YjeQ is a protein broadly conserved in bacteria containing an N-terminal oligonucleotide/oligosaccharide fold (OB-fold) domain, a central GTPase domain, and a C-terminal zinc-finger domain. YjeQ binds tightly and stoichiometrically to the 30S subunit, which stimulates its GTPase activity by 160-fold. Despite growing evidence for the involvement of the YjeQ protein in bacterial 30S subunit assembly, the specific function and mechanism of this protein remain unclear. Here, we report the costructure of YjeQ with the 30S subunit obtained by cryo-electron microscopy. The costructure revealed that YjeQ interacts simultaneously with helix 44, the head and the platform of the 30S subunit. This binding location of YjeQ in the 30S subunit suggests a chaperone role in processing of the 3' end of the rRNA as well as in mediating the correct orientation of the main domains of the 30S subunit. In addition, the YjeQ binding site partially overlaps with the interaction site of initiation factors 2 and 3, and upon binding, YjeQ covers three inter-subunit bridges that are important for the association of the 30S and 50S subunits. Hence, our structure suggests that YjeQ may assist in ribosome maturation by preventing premature formation of the translation initiation complex and association with the 50S subunit. Together, these results support a role for YjeQ in the late stages of 30S maturation.     

Conserved P-loop GTPases of unknown function in bacteria: an emerging and vital ensemble in bacterial physiology.

Establishing the roles of conserved gene products in bacteria is of fundamental importance to our understanding of the core protein complement necessary to sustain cellular life. P-loop GTPases and related ATPases represent an abundant and remarkable group of proteins in bacteria that, in many cases, have evaded characterization. Here, efforts aimed at understanding the cellular function of a group of 8 conserved, poorly characterized genes encoding P-loop GTPases, era, obg, trmE, yjeQ, engA, yihA, hflX, ychF, and a related ATPase, yjeE, are reviewed in considerable detail. While concrete cellular roles remain elusive for all of these genes and considerable pleiotropy has plagued their study, experiments to date have frequently implicated the ribosome. In the case of era, obg, yjeQ, and engA, the evidence is most consistent with roles in ribosome biogenesis, though the prediction is necessarily putative. While the protein encoded in trmE clearly has a catalytic function in tRNA modification, the participation of its GTPase domain remains obscure, as do the functions of the remaining proteins. A full understanding of the cellular functions of all of these important proteins remains the goal of ongoing studies of cellular phenotype and protein biochemistry.     

A Bacterial GAP-Like Protein, YihI, Regulating the GTPase of Der, an Essential GTP-Binding Protein in Escherichia coli

Der (double Era-like GTPase) is an essential GTPase consisting of two GTP-binding motifs in tandem followed by a KH-like domain. Der plays a critical role in 50S ribosome maturation at a later biogenesis step. Here, we attempted to identify a protein interacting with Der that modulates its function and regulation. Using a yeast two-hybrid, we discovered that Der interacts with YihI, which activates the GTPase activity of Der. Its overexpression affected cell growth, causing accumulation of rRNA precursors and an aberrant ribosome profile that was similar to that of Der-depleted cells, suggesting that Der and YihI are involved in the 50S ribosome assembly. The yihI deletion strain showed a shorter lag phase than wild-type strain, suggesting that YihI may be a negative regulator for ribosome assembly. We propose that YihI is a GAP (GTPase-activating protein)-like protein that modulates Der function to negatively regulate cell growth at the beginning of exponential growth.     

The C-terminal helix in the YjeQ zinc-finger domain catalyzes the release of RbfA during 30S ribosome subunit assembly

YjeQ (also called RsgA) and RbfA proteins in Escherichia coli bind to immature 30S ribosome subunits at late stages of assembly to assist folding of the decoding center. A key step for the subunit to enter the pool of actively translating ribosomes is the release of these factors. YjeQ promotes dissociation of RbfA during the final stages of maturation; however, the mechanism implementing this functional interplay has not been elucidated. YjeQ features an amino-terminal oligonucleotide/oligosaccharide binding domain, a central GTPase module and a carboxy-terminal zinc-finger domain. We found that the zinc-finger domain is comprised of two functional motifs: the region coordinating the zinc ion and a carboxy-terminal α-helix. The first motif is essential for the anchoring of YjeQ to the 30S subunit and the carboxy-terminal α-helix facilitates the removal of RbfA once the 30S subunit reaches the mature state. Furthermore, the ability of the mature 30S subunit to stimulate YjeQ GTPase activity also depends on the carboxy-terminal α-helix. Our data are consistent with a model in which YjeQ uses this carboxy-terminal α-helix as a sensor to gauge the conformation of helix 44, an essential motif of the decoding center. According to this model, the mature conformation of helix 44 is sensed by the carboxy-terminal α-helix, which in turn stimulates the YjeQ GTPase activity. Hydrolysis of GTP is believed to assist the release of YjeQ from the mature 30S subunit through a still uncharacterized mechanism. These results identify the structural determinants in YjeQ that implement the functional interplay with RbfA.     

YjeQ,an essential,conserved, uncharacterized protein from Escherichia coli,is an unusual GTPase with circularly permuted G-motifs and marked burst kinetics

The Escherichia coli protein YjeQ represents a protein family whose members are broadly conserved in bacteria and have been shown to be indispensable to the growth of E. coli and Bacillus subtilis [Arigoni, F., et al. (1998) Nat. Biotechnol. 16, 851]. Proteins of the YjeQ family contain all sequence motifs typical of the vast class of P-loop-containing GTPases, but show a circular permutation, with a G4-G1-G3 pattern of motifs as opposed to the regular G1-G3-G4 pattern seen in most GTPases. All YjeQ family proteins display a unique domain architecture, which includes a predicted N-terminal OB-fold RNA-binding domain, the central permuted GTPase module, and a zinc knuckle-like C-terminal cysteine cluster. This domain architecture suggests a possible role for YjeQ as a regulator of translation. YjeQ was overexpressed, purified to homogeneity, and shown to contain 0.6 equiv of GDP. Steady state kinetic analyses indicated slow GTP hydrolysis, with a k(cat) of 9.4 h(-)(1) and a K(m) for GTP of 120 microM (k(cat)/K(m) = 21.7 M(-)(1) s(-)(1)). YjeQ also hydrolyzed other nucleoside triphosphates and deoxynucleotide triphosphates such as ATP, ITP, and CTP with specificity constants (k(cat)/K(m)) ranging from 0.2 to 1.0 M(-)(1) s(-)(1). Pre-steady state kinetic analysis of YjeQ revealed a burst of nucleotide hydrolysis for GTP described by a first-order rate constant of 100 s(-)(1) as compared to a burst rate of 0.2 s(-)(1) for ATP. In addition, a variant in the G1 motif of YjeQ (S221A) was substantially impaired for GTP hydrolysis (0.3 s(-)(1)) with a less significant impact on the steady state rate (1.8 h(-)(1)). In summary, E. coli YjeQ is an unusual, circularly permuted P-loop-containing GTPase, which catalyzes GTP hydrolysis at a rate 45 000 times greater than that of turnover.     

The Escherichia coli GTPase CgtAE is involved in late steps of large ribosome assembly

The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which is poorly understood. Although several bona fide assembly factors have been identified, their precise functions and temporal relationships are not clearly defined. Here we describe the involvement of an Escherichia coli GTPase, CgtA(E), in late steps of large ribosomal subunit biogenesis. CgtA(E) belongs to the Obg/CgtA GTPase subfamily, whose highly conserved members are predominantly involved in ribosome function. Mutations in CgtA(E) cause both polysome and rRNA processing defects; small- and large-subunit precursor rRNAs accumulate in a cgtA(E) mutant. In this study we apply a new semiquantitative proteomic approach to show that CgtA(E) is required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribosomal subunit. Moreover, we demonstrate the interaction with the 50S ribosomal subunits of specific nonribosomal proteins (including heretofore uncharacterized proteins) and define possible temporal relationships between these proteins and CgtA(E). We also show that purified CgtA(E) associates with purified ribosomal particles in the GTP-bound form. Finally, CgtA(E) cofractionates with the mature 50S but not with intermediate particles accumulated in other large ribosome assembly mutants.     

Suppression of a cold-sensitive mutation in ribosomal protein S5 reveals a role for RimJ in ribosome biogenesis

A specific mutation of Escherichia coli ribosomal protein S5, in which glycine is changed to aspartate at position 28 [S5(G28D)], results in cold sensitivity and defects in ribosome biogenesis and translational fidelity. In an attempt to understand the roles of S5 in these essential cellular functions, we selected extragenic suppressors and identified rimJ as a high-copy suppressor of the cold-sensitive phenotype associated with the S5(G28D) mutation. Our studies indicate that RimJ overexpression suppresses the growth defects, anomalous ribosome profiles and mRNA misreading exhibited by the S5(G28D) mutant strain. Although previously characterized as the N -acetyltransferase of S5, our data indicate that RimJ, when devoid of acetyltransferase activity, can suppress S5(G28D) defects thus indicating that the suppression activity of RimJ is not dependent on its acetyltransferase activity. Additionally, RimJ appears to associate with pre-30S subunits indicating that it acts on the ribonucleoprotein particle. These findings suggest that RimJ has evolved dual functionality; it functions in r-protein acetylation and as a ribosome assembly factor in E. coli .     

Inactivation of the RluD pseudouridine synthase has minimal effects on growth and ribosome function in wild-type Escherichia coli and Salmonella enterica

The Escherichia coli rluD gene encodes a pseudouridine synthase responsible for the pseudouridine (Ψ) modifications at positions 1911, 1915, and 1917 in helix 69 of 23S rRNA. It has been reported that deletion of rluD in K-12 strains of E. coli is associated with extremely slow growth, increased readthrough of stop codons, and defects in 50S ribosomal subunit assembly and 30S-50S subunit association. Suppressor mutations in the prfB and prfC genes encoding release factor 2 (RF2) and RF3 that restore the wild type-growth rate and also correct the ribosomal defects have now been isolated. These suppressors link helix 69 Ψ residues with the termination phase of protein synthesis. However, further genetic analysis reported here also reveals that the slow growth and other defects associated with inactivation of rluD in E. coli K-12 strains are due to a defective RF2 protein, with a threonine at position 246, which is present in all K-12 strains. This is in contrast to the more typical alanine found at this position in most bacterial RF2s, including those of other E. coli strains. Inactivation of rluD in E. coli strains containing the prfB allele from E. coli B or in Salmonella enterica, both carrying an RF2 with Ala246, has negligible effects on growth, termination, or ribosome function. The results indicate that, in contrast to those in wild bacteria, termination functions in E. coli K-12 strains carrying a partially defective RF2 protein are especially susceptible to perturbation of ribosome-RF interactions, such as that caused by loss of h69 Ψ modifications.     

Up-regulation of yggG promotes the survival of Escherichia coli cells containing Era-1 mutant protein

Era is a highly conserved GTPase essential for bacterial growth. Using a digoxigenin-labeled Era protein to screen a phage expression library of Escherichia coli genomic DNA, yggG, a gene that encodes a putative zinc metalloprotease was isolated and characterized. The deduced amino acid sequence of YggG showed high degrees of similarity to some reported heat shock proteins. In this study, the direct interaction between Era and YggG was confirmed, and it was found that the yggG gene, encoding a 25 kDa heat shock protein, was up-regulated at the mRNA level in partially defective Era GTPase mutants (era-1) and in E. coli cells overproducing Era-1. The delta yggG strain displayed the same growth rate as wild-type strain under normal growth conditions and after heat shock. Overexpression of Era-1 in the delta yggG strain resulted in a stronger growth-inhibitory effect than that in the wild-type strain, while coexpression of YggG partially restored the bacterial growth rate. The results indicated that YggG expression is significantly increased in response to stress caused by the Era-1 mutant protein in E. coli, thus promoting the growth of E. coli.     

Overexpression of two different GTPases rescues a null mutation in a heat-induced rRNA methyltransferase

The Escherichia coli RrmJ (FtsJ) heat shock protein functions as an rRNA methyltransferase that modifies position U2552 of 23S rRNA in intact 50S ribosomal subunits. An in-frame deletion of the rrmJ (ftsJ) gene leads to severe growth disadvantages under all temperatures tested and causes significant accumulation of ribosomal subunits at the expense of functional 70S ribosomes. To investigate whether overexpression of other E. coli genes can restore the severe growth defect observed in rrmJ null mutants, we constructed an overexpression library from the rrmJ deletion strain and cloned and identified the E. coli genes that were capable of rescuing the rrmJ mutant phenotype. Our intention was to identify other methylases whose specificities overlapped enough with that of RrmJ to allow complementation when overexpressed. To our great surprise, no methylases were found by this method; rather, two small GTPases, Obg (YhbZ) and EngA, when overexpressed in the rrmJ deletion strains, were found to restore the otherwise severely impaired ribosome assembly process and/or stability of 70S ribosomes. 50S ribosomal subunits prepared from these overexpressing strains were shown to still serve as in vitro substrates for purified RrmJ, indicating that the 23S rRNA likely was still lacking the highly conserved Um2552 modification. The apparent lack of this modification, however, no longer caused ribosome defects or a growth disadvantage. Massive overexpression of another related small GTPase, Era, failed to rescue the growth defects of an rrmJ strain. These findings suggest a hitherto unexpected connection between rRNA methylation and GTPase function, specifically that of the two small GTPases Obg and EngA.     

RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis

RsgA is a 30S ribosomal subunit-binding GTPase with an unknown function, shortage of which impairs maturation of the 30S subunit. We identified multiple gain-of-function mutants of Escherichia coli rbfA, the gene for a ribosome-binding factor, that suppress defects in growth and maturation of the 30S subunit of an rsgA-null strain. These mutations promote spontaneous release of RbfA from the 30S subunit, indicating that cellular disorders upon depletion of RsgA are due to prolonged retention of RbfA on the 30S subunit. We also found that RsgA enhances release of RbfA from the mature 30S subunit in a GTP-dependent manner but not from a precursor form of the 30S subunit. These findings indicate that the function of RsgA is to release RbfA from the 30S subunit during a late stage of ribosome biosynthesis. This is the first example of the action of a GTPase on the bacterial ribosome assembly described at the molecular level.     

Mutations in the gene for EF-G reduce the requirement for 4.5S RNA in the growth of E. coli

A general strategy is described for the isolation of suppressors of essential genes whose functions are unknown. This strategy was used to analyze the role of 4.5S RNA, an essential RNA of E. coli. In this strategy, the structural gene for 4.5S RNA is fused to the Ptac promoter in such a way that the strain becomes dependent upon inducers of lac for growth. Mutants mapping to fus, the structural gene for protein synthesis elongation factor G, appear as spontaneous, inducer-independent revertants. These mutants alter the intracellular distribution of 4.5S RNA such that it sediments at 70S or greater. Furthermore, the increased sedimentation velocity is sensitive to the antibiotic puromycin. These results show that 4.5S RNA physically associates with the ribosome in performing its essential function, and that this association is mediated by elongation factor G.     

Salmonella enterica serovar Typhimurium BipA exhibits two distinct ribosome binding modes

BipA is a highly conserved prokaryotic GTPase that functions to influence numerous cellular processes in bacteria. In Escherichia coli and Salmonella enterica serovar Typhimurium, BipA has been implicated in controlling bacterial motility, modulating attachment and effacement processes, and upregulating the expression of virulence genes and is also responsible for avoidance of host defense mechanisms. In addition, BipA is thought to be involved in bacterial stress responses, such as those associated with virulence, temperature, and symbiosis. Thus, BipA is necessary for securing bacterial survival and successful invasion of the host. Steady-state kinetic analysis and pelleting assays were used to assess the GTPase and ribosome-binding properties of S. enterica BipA. Under normal bacterial growth, BipA associates with the ribosome in the GTP-bound state. However, using sucrose density gradients, we demonstrate that the association of BipA and the ribosome is altered under stress conditions in bacteria similar to those experienced during virulence. The data show that this differential binding is brought about by the presence of ppGpp, an alarmone that signals the onset of stress-related events in bacteria.     

Changes in ribosome biogenesis may induce cancer by down-regulating the cell tumor suppressor potential

Many human pathological conditions, not linked to genetic alterations of oncogenes or tumor suppressors, are nevertheless associated with an increased risk of developing cancer, and some of them are characterized by quantitative and/or qualitative changes in ribosome biogenesis. Indeed, there is evidence that both an up-regulation of ribosome biogenesis, such as that occurring during the abnormal stimulation of cell growth, and intrinsic dysfunctions of ribosomes, such as those characterizing a series of inherited disorders, show an increased incidence of tumor onset. Here we discuss some recent insights into the mechanisms by which these alterations in ribosome biogenesis may facilitate tumorigenesis.     

Functional Escherichia coli 23S rRNAs containing processed and unprocessed intervening sequences from Salmonella typhimurium.

We have introduced the intervening sequence (IVS) from 23S rRNA of the rrnD operon of Salmonella typhimurium into the equivalent position of Escherichia coli 23S rRNA. Salmonella typhimurium 23S rRNA is fragmented due to the RNase III-dependent removal of the approximately 100 nt stem-loop structure that comprises the IVS. In this study, we have found that insertion of the S. typhimurium IVS into E. coli 23S rRNA causes fragmentation of the RNA but does not affect ribosome function. Cells expressing the fragmented 23S rRNA exhibited wild-type growth rates. Fragmented RNA was found in the actively translating polysome pool and did not alter the sedimentation profile of ribosomal subunits, 70S ribosomes or polysomes. Finally, hybrid 23S rRNA carrying the A2058G mutation conferred high level erythromycin resistance indistinguishable from that of intact 23S rRNA carrying this mutation. These observations indicate that the presence of this IVS and its removal are phenotypically silent. As observed in an RNase III-deficient strain, processing of the IVS was not required for the production of functional ribosomes.     

Conformationally restricted elongation factor G retains GTPase activity but is inactive in translocation on the ribosome

Elongation factor G (EF-G) from Escherichia coli is a large, five-domain GTPase that promotes tRNA translocation on the ribosome. Full activity requires GTP hydrolysis, suggesting that a conformational change of the factor is important for function. To restrict the intramolecular mobility, two cysteine residues were engineered into domains 1 and 5 of EF-G that spontaneously formed a disulfide cross-link. Cross-linked EF-G retained GTPase activity on the ribosome, whereas it was inactive in translocation as well as in turnover. Both activities were restored when the cross-link was reversed by reduction. These results strongly argue against a GTPase switch-type model of EF-G function and demonstrate that conformational mobility is an absolute requirement for EF-G function on the ribosome.