The tandem GTPase, Der, is essential for the biogenesis of 50S ribosomal subunits in Escherichia coli

Abstract

SummaryA unique GTP-binding protein, Der contains two consecutive GTP-binding domains at the N-terminal region and its homologues are highly conserved in eubacteria but not in archaea and eukaryotes. In the present paper, we demonstrate that Der is one of the essential GTPases in Escherichia coli and that the growth rate correlates with the amount of Der in the cell. Interestingly, both GTP-binding domains are required at low temperature for cell growth, while at high temperature either one of the two domains is dispensable. Result of the sucrose density gradient experiment suggests that Der interacts specifically with 50S ribosomal subunits only in the presence of a GTP analogue, GMPPNP. The depletion of Der accumulates 50S and 30S ribosomal subunits with a concomitant reduction of polysomes and 70S ribosomes. Notably, Der-depleted cells accumulate precursors of both 23S and 16S rRNAs. Moreover, at lower Mg 2+ concentration, 50S ribosomal subunits from Derdepleted cells are further dissociated into aberrant 50S ribosomal subunits; however, 30S subunits are stable. It was revealed that the aberrant 50S subunits, 40S subunits, contain less ribosomal proteins with significantly reduced amounts of L9 and L18. These results suggest that Der is a novel 50S ribosomeassociated factor involved in the biogenesis and stability of 50S ribosomal subunits. We propose that Der plays a pivotal role in ribosome biogenesis possibly through interaction with rRNA or rRNA/r-protein complex.

Conserved GTPase LepA (Elongation Factor 4) functions in biogenesis of the 30S subunit of the 70S ribosome

“…Based on these and additional data, the authors propose that the final stages of ribosome maturation take place in conjunction with its first round of initiation. This model can rationalize several earlier observations, including the presence of precursor rRNAs in polysomes (36,37), links between IF2 and ribosome assembly (12,38), and inhibition of 16S rRNA processing resulting from defects in 50S assembly (39,40).…”

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

“…[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.…”

“…13,14 In case of T. maritima Der, each of the two Der GTP-binding domains expressed separately has an intrinsic GTPase activity, though the activity of the N-terminal GD1 domain is stronger than that of the GD2 domain. 13 It has been demonstrated that Der has a critical role in 50S subunit biogenesis, as it associates with 50S subunit in the presence of a GTP analog 9,15 and its depletion caused conformational instability of 50S subunit at a low Mg 2+ concentration. 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.…”

“…13 It has been demonstrated that Der has a critical role in 50S subunit biogenesis, as it associates with 50S subunit in the presence of a GTP analog 9,15 and its depletion caused conformational instability of 50S subunit at a low Mg 2+ concentration. 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.…”

“…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.…”