Interaction of an Essential Escherichia coli GTPase, Der, with the 50S Ribosome via the KH-Like Domain

Abstract

Der, an essential Escherichia coli tandem GTPase, has been implicated in 50S subunit biogenesis. The rrmJ gene encodes a methyltransferase that modifies the U2552 residue of 23S rRNA, and its deletion causes a severe growth defect. Peculiarly, overexpression of Der suppresses growth impairment. In this study, using an rrmJ-deletion strain, we demonstrated that two GTPase domains of Der regulate its association with 50S subunit via the KH-like domain. We also identified a region of Der that is critical for its specific interaction with 50S subunit. Emerging evidence indicates that many Escherichia coliGTPases play critical roles in ribosome biogenesis (6). For example, E. coli Era consists of a conventional GTP-binding domain and a KH domain (an RNA-binding domain) with a consensus VIGXXGXXI sequence (9). The direct interaction between Era and 16S rRNA was demonstrated by structural studies of a Thermus thermophilus 30S ribosomal subunit complexed with Era (24). Peculiarly, Era was shown to suppress the cold-sensitive cell growth of the rbfA-deletion strain whose gene product resembles a KH domain and plays an important role in 30S subunit assembly at low temperature (13, 16). A unique GTPase subfamily of Der (double Era-like GTPase; also known as EngA) is conserved only in eubacteria. We have previously demonstrated that Der is cofractionated with 50S subunits in a GTP-dependent manner and that Der plays a critical role in 50S subunit maturation at a later biogenesis step (15).Interestingly, both GTP-binding domains (G domains) were essential for cell growth; moreover, the two G domains function cooperatively, suggesting that GTP-induced conformational changes and GTPase activity are essential for cell viability as well as function (1, 15). The X-ray crystal structures of two Der orthologs from Thermotoga maritima and Bacillus subtilis revealed that the C-terminal domain has a topology similar to that of a KH domain without a consensus sequence motif and is flanked by two G domains (22, 23). It was suggested that the GTP-bound form of YphC (a Der ortholog in B. subtilis) triggers a dramatic conformational change, which favors an interaction with negatively charged ribonucleic acids by exposing a positively charged KH-like domain with a high pI value (14, 22).Overexpression of E. coli Der functionally suppressed the slow growth defect of a deletion strain of the rrmJ gene, whose gene product is a methyltransferase, modifying the U2552 residue in the A loop of 23S rRNA in an intact 50S subunit (5, 26). Even though ⌬rrmJ (strain HB23) is viable, it causes a serious defect of cell growth by accumulating 50S and 30S ribosomal subunits at the expense of 70S ribosomes. Thus, overexpression of Der seems to overcome its weak interaction with 50S subunits that are unmethylated at U2552. In this study, using an ⌬rrmJ strain as a genetic background, we tried to elucidate the nature of the functional regulation of two G domains and the KH-like domain. We further characterized the KH-like domain by random mutagenesis and iden...

Crippling the Essential GTPase Der Causes Dependence on Ribosomal Protein L9

“…Part of the conundrum stems from the fact that we do not know what Der specifically does. Der depletion for extended periods causes the accumulation of unstable and/or incomplete large subunits and defects in both 16S and 23S rRNA processing, which others have suggested points to a role in ribosome biogenesis (23)(24)(25)(26)51). Cells with deficient rRNA folding factors commonly display cold sensitivities (46,52).…”

“…Here, we describe strains with mutations in the essential ribosome biogenesis GTPase Der (EngA/YphC) that grow better with L9 than without it (23,24). We show that the L9-dependent Der mutants are severely compromised for GTPase activity, which was previously shown to be critical for Der's essential function (23,25,26). Purified L9 does not rescue the GTPase defects, nor does it alter the stimulatory activity of Der's GAP-like factor YihI, so the fitness afforded by L9 seems indirect.…”

Structure-based design and screening of inhibitors for an essential bacterial GTPase, Der

“…9 GTP binds to Der in a cooperative manner and the interruption of cooperative nucleotide association disrupts the interaction of Der with the 50S subunit. 11,12,15,16 The essential role of Der in 50S subunit assembly was further supported by the isolation of Der as a suppressor for the rrmJ null phenotype. 17 RrmJ is a 23S ribosomal RNA methyltransferase and its deletion resulted in reduction of the functional 70S ribosomes and accumulation of 30S and 50S subunits.…”

“…[4][5][6] Even though there is low primary sequence similarity among different GTPase families, most GTPases have an impressively common structural topology consisting of six b-strands surrounded by five a-helices. 4,5,7 Previously we characterized an essential GTPase, Der (double Eralike protein), from Escherichia coli (EcDer) [8][9][10][11][12] and from a hyperthermophilic bacterium Thermotoga maritima (TmDer). 8,13 The 3D structure of TmDer was solved by X-ray crystallography.…”

“…The accumulated 50S subunits in rrmJ deletion cells were also disintegrated into abnormal subunits at a lower Mg 2+ concentration similar to that observed in the Der-depleted cells. 9,11,17 Furthermore, the primary sequence of Der is highly ubiquitous in most eubacteria. Importantly, this GTPase subfamily does not exist in eukaryotes.…”