Isolation and Characterization of a Dominant Negative Mutant of Bacillus subtilis GTP-binding Protein, YlqF, Essential for Biogenesis and Maintenance of the 50 S Ribosomal Subunit

Matsuo, Yoshitaka; Oshima, Taku; Loh, Pek Chin; Morimoto, Takahiro; Ogasawara, Nagahisa

doi:10.1074/jbc.m703894200

Cited by 24 publications

(27 citation statements)

References 38 publications

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“…RbgA/YlqF is an essential GTPase required for the proliferation of B. subtilis (36,37). Depletion of RbgA or expression of a dominant-negative mutant of RbgA resulted in accumulation of the 45S particles (36)(37)(38)(39). However, unlike the E. coli 45S precursor, the B. subtilis 45S particle does not alter its sedimentation coefficient, even at 10 mM Mg 2+ (36,39).…”

Section: Discussionmentioning

confidence: 99%

“…Depletion of RbgA or expression of a dominant-negative mutant of RbgA resulted in accumulation of the 45S particles (36)(37)(38)(39). However, unlike the E. coli 45S precursor, the B. subtilis 45S particle does not alter its sedimentation coefficient, even at 10 mM Mg 2+ (36,39). In addition, there is no homolog of RbgA in E. coli (10), and no homolog of RlmE in B. subtilis; consistent with this knowledge, U2552 in 23S rRNA remains unmethylated in B. subtilis (40).…”

Section: Discussionmentioning

confidence: 99%

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Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly

Arai

Ishiguro

Kimura

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Ribosome biogenesis requires multiple assembly factors. In Escherichia coli, deletion of RlmE, the methyltransferase responsible for the 2′-O-methyluridine modification at position 2552 (Um2552) in helix 92 of the 23S rRNA, results in slow growth and accumulation of the 45S particle. We demonstrate that the 45S particle that accumulates in ΔrlmE is a genuine precursor that can be assembled into the 50S subunit. Indeed, 50S formation from the 45S precursor could be promoted by RlmE-mediated Um2552 formation in vitro. Ribosomal protein L36 (encoded by rpmJ) was completely absent from the 45S precursor in ΔrlmE, and we observed a strong genetic interaction between rlmE and rpmJ. Structural probing of 23S rRNA and high-salt stripping of 45S components revealed that RlmE-mediated methylation promotes interdomain interactions via the association between helices 92 and 71, stabilized by the single 2′-O-methylation of Um2552, in concert with the incorporation of L36, triggering late steps of 50S subunit assembly.R ibosomes are essential ribonucleoprotein complexes that translate the genetic information encoded by mRNA into protein. Intact bacterial ribosomes, which have a sedimentation coefficient of 70S, consist of large (50S) and small (30S) subunits. Each subunit can be reconstituted in vitro from its individual component ribosomal RNAs and proteins (1-5), indicating that these components contain all of the information necessary to automatically assemble into functional subunits. Because the intermediate particles observed in an in vitro reconstitution of the subunits are similar to those observed in vivo, assembly maps that describe the order of ribosomal protein binding in vitro are useful tools for understanding ribosome biogenesis in the cell (6). However, assembly is slower in vitro than in vivo, and nonphysiological conditions, such as high-salt concentration and high temperature, are required for in vitro assembly.In the cell, ribosome assembly is coordinated with the transcription and processing of large precursor rRNAs encoded by the rrn operon (3, 7). Once transcription starts, nascent transcripts rapidly form local secondary and tertiary structures that serve as binding sites for the primary ribosomal proteins, thereby initiating ribosome assembly. However, hierarchical assembly starting at the 5′-terminal domains of the 16S and 23S rRNAs is not essential for ribosome formation in the cell (8) because nonribosomal factors, termed "assembly factors," facilitate rapid and efficient assembly of ribosomal particles. Ribosome biogenesis is an energy-consuming process in which a number of assembly factors, including RNA helicases, GTPases, protein chaperones, and rRNA-modifying enzymes, support efficient assembly of each subunit (6, 9, 10). Individual mutation or deletion of several assembly factors causes accumulation of immature 50S subunits that sediment at 40S or 45S. For example, the RNA helicase SrmB participates in the early stage of 50S assembly (11,12); deletion of srmB results in accumulation of a 40S ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly

Arai

Ishiguro

Kimura

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…In fact, it has been shown that the equivalent segment in B. subtilis YlqF (Fig. S6), another circularly permutated GTPase involved in the 50S subunit assembly (27), is important in the nucleotide modulation of the binding of YlqF to the 50S subunit (28).…”

Section: Resultsmentioning

confidence: 99%

Structural basis for the function of a small GTPase RsgA on the 30S ribosomal subunit maturation revealed by cryoelectron microscopy

Guo¹,

Yuan²,

Xu³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The bacterial RsgA, a circularly permutated GTPase, whose GTPase activity is dependent on the 30S ribosomal subunit, is a late-stage ribosome biogenesis factor involved in the 30S subunit maturation. The role of RsgA is to release another 30S biogenesis factor, RbfA, from the mature 30S subunit in a GTP-dependent manner. Using cryoelectron microscopy, we have determined the structure of the 30S subunit bound with RsgA in the presence of GMPPNP at subnanometer resolution. In the structure, RsgA binds to the central part of the 30S subunit, close to the decoding center, in a position that is incompatible with multiple biogenesis factors, all three translation initiation factors, as well as A-, P-site tRNAs and the 50S subunit. Further structural analysis not only provides a structural model for the RsgA-dependent release of RbfA from the nascent 30S subunit, but also indicates RsgA's role in the ribosomal protein assembly, to promote some tertiary binding protein incorporation. Moreover, together with available biochemical and genetic data, our results suggest that RsgA might be a general checkpoint protein in the late stage of the 30S subunit biogenesis, whose function is not only to release biogenesis factors (e.g., RbfA) from the nascent 30S subunit, but also to block the association of initiation factors to the premature 30S subunit. YjeQ | single-particle reconstruction | molecular dynamic flexible fitting

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“…These results suggest that OsNug2 is a GTP/GDP-binding protein that is comparable with that of other YawG subfamily proteins. Interestingly, a few recent studies have shown that some GTPases play important roles in the ribosomal maturation process and that their GTPase activities are enhanced in the presence of ribosomal subunits in vitro (21,25,48). In light of these observations, we measured the kinetic parameters of OsNug2 hydrolytic activity in the presence 40S, 60S, or the 80S ribosome purified from rice.…”

Section: Ubiquitous Expression Of Two Plant Nug2 Genes Belonging To Tmentioning

confidence: 99%

Nuclear/Nucleolar GTPase 2 Proteins as a Subfamily of YlqF/YawG GTPases Function in Pre-60S Ribosomal Subunit Maturation of Mono- and Dicotyledonous Plants

Hwang

Son

et al. 2011

Journal of Biological Chemistry

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The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with k cat values 0.12 ؎ 0.007 min ؊1 (n ‫؍‬ 6) and 0.087 ؎ 0.002 min ؊1 (n ‫؍‬ 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S prerRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono-and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm.In eukaryotes, ribosome biogenesis is a complex and coordinated process that involves the synthesis and processing of rRNA, and its assembly with ribosomal proteins (1, 2). Ribosome biogenesis begins with transcription of rDNA repeats by RNA polymerase I and III, followed by processing and assembly of pre-rRNA intermediates, which are exported to the cytoplasm from the nucleolus and nucleus (3-9). These processes require various ribosome biogenesis factors such as assembly factors, as well as non-ribosomal factors including snoRNPs, endo-and exonucleases, pseudouridine synthases, methyltransferases, RNA helicases, RNA chaperones, ATPases and GTPases (3,4,[6][7][8]10).Several GTPases are essential for the biogenesis and assembly of ribosomal subunits (1, 10 -13). In Escherichia coli, the process of the 50S ribosomal subunit assembly requires Obg, EngA, and HflX (14 -16), whereas Bacillus subtilis needs . Interestingly, most GTPases involved in ribosomal biogenesis belong to the YlqF/YawG family, which is characterized by a circularly permuted order of GTPase motifs within the GTP-binding domain (1,11,20). In E. coli, YjeQ binds to the 30S ribosomal subunit, whereas YloQ and YqeH show similar activities in B. subtilis (21-23). YlqF (RbgA) participates in late stage assembly of the 50...

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Isolation and Characterization of a Dominant Negative Mutant of Bacillus subtilis GTP-binding Protein, YlqF, Essential for Biogenesis and Maintenance of the 50 S Ribosomal Subunit

Cited by 24 publications

References 38 publications

Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly

Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly

Structural basis for the function of a small GTPase RsgA on the 30S ribosomal subunit maturation revealed by cryoelectron microscopy

Nuclear/Nucleolar GTPase 2 Proteins as a Subfamily of YlqF/YawG GTPases Function in Pre-60S Ribosomal Subunit Maturation of Mono- and Dicotyledonous Plants

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