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

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.

Download Full-text

Genetic Interaction Screens with Ordered Overexpression and Deletion Clone Sets Implicate the Escherichia coli GTPase YjeQ in Late Ribosome Biogenesis

Journal of Bacteriology ◽

10.1128/jb.01744-07 ◽

2008 ◽

Vol 190 (7) ◽

pp. 2537-2545 ◽

Cited By ~ 38

Author(s):

Tracey L. Campbell ◽

Eric D. Brown

Keyword(s):

Escherichia Coli ◽

Ribosome Biogenesis ◽

Slow Growth ◽

Genetic Interaction ◽

Initiation Factor ◽

Genetic Screens ◽

Genomic Libraries ◽

Initiation Factor 2 ◽

Bona Fide

ABSTRACT The Escherichia coli protein YjeQ is a circularly permuted GTPase that is broadly conserved in bacteria. An emerging body of evidence, including cofractionation and in vitro binding to the ribosome, altered polysome profiles after YjeQ depletion, and stimulation of GTPase activity by ribosomes, suggests that YjeQ is involved in ribosome function. The growth of strains lacking YjeQ in culture is severely compromised. Here, we probed the cellular function of YjeQ with genetic screens of ordered E. coli genomic libraries for suppressors and enhancers of the slow-growth phenotype of a ΔyjeQ strain. Screening for suppressors using an ordered library of 374 clones overexpressing essential genes and genes associated with ribosome function revealed that two GTPases, Era and initiation factor 2, ameliorated the growth and polysome defects of the ΔyjeQ strain. In addition, seven bona fide enhancers of slow growth were identified (Δtgt, ΔksgA, ΔssrA, ΔrimM, ΔrluD, ΔtrmE/mnmE, and ΔtrmU/mnmA) among 39 deletions (in genes associated with ribosome function) that we constructed in the ΔyjeQ genetic background. Taken in context, our work is most consistent with the hypothesis that YjeQ has a role in late 30S subunit biogenesis.

Download Full-text

The Loop 2 Region of Ribosomal Protein uS5 Influences Spectinomycin Sensitivity, Translational Fidelity, and Ribosome Biogenesis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01186-16 ◽

2016 ◽

Vol 61 (2) ◽

Cited By ~ 3

Author(s):

Divya Kamath ◽

Steven T. Gregory ◽

Michael O'Connor

Keyword(s):

Escherichia Coli ◽

Ribosomal Protein ◽

Recent Work ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Rrna Processing ◽

Translational Fidelity ◽

Subunit Assembly ◽

Essential Component ◽

Loop 2

ABSTRACT Ribosomal protein uS5 is an essential component of the small ribosomal subunit that is involved in subunit assembly, maintenance of translational fidelity, and the ribosome's response to the antibiotic spectinomycin. While many of the characterized uS5 mutations that affect decoding map to its interface with uS4, more recent work has shown that residues distant from the uS4-uS5 interface can also affect the decoding process. We targeted one such interface-remote area, the loop 2 region (residues 20 to 31), for mutagenesis in Escherichia. coli and generated 21 unique mutants. A majority of the loop 2 alterations confer resistance to spectinomycin and affect the fidelity of translation. However, only a minority show altered rRNA processing or ribosome biogenesis defects.

Download Full-text

Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

Nature Communications ◽

10.1038/s41467-021-23702-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hauke S. Hillen ◽

Elena Lavdovskaia ◽

Franziska Nadler ◽

Elisa Hanitsch ◽

Andreas Linden ◽

...

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Structural Basis ◽

Peptidyl Transferase ◽

Mitochondrial Ribosomes ◽

Mitochondrial Ribosome ◽

In Vitro Reconstitution

AbstractRibosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

Download Full-text

An eIF-4A-like protein is a suppressor of an Escherichia coli mutant defective in 50S ribosomal subunit assembly

Nature ◽

10.1038/336496a0 ◽

1988 ◽

Vol 336 (6198) ◽

pp. 496-498 ◽

Cited By ~ 91

Author(s):

Kayoko Nishi ◽

Francoise Morel-Deville ◽

John W. B. Hershey ◽

Terrance Leighton ◽

Joachim Schnier

Keyword(s):

Escherichia Coli ◽

Ribosomal Subunit ◽

Subunit Assembly ◽

Coli Mutant

Download Full-text

In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits

Biochemical Journal ◽

10.1042/bj20060038 ◽

2006 ◽

Vol 396 (3) ◽

pp. 565-571 ◽

Cited By ~ 28

Author(s):

Takaomi Nomura ◽

Kohji Nakano ◽

Yasushi Maki ◽

Takao Naganuma ◽

Takashi Nakashima ◽

...

Keyword(s):

Escherichia Coli ◽

Ribosomal Proteins ◽

Synthetic Activity ◽

23S Rrna ◽

Elongation Factors ◽

Hybrid Form ◽

Pyrococcus Horikoshii ◽

E Coli ◽

Functional Features

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0·Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0·Ph-L12 complex and Ph-L11 could replace L10·L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.

Download Full-text

A comparison of the unfolding and dissociation of the large ribosome subunits from Rhodopseudomonas·spheroides N.C.I.B. 8253 and Escherichia coli M.R.E. 600

Biochemical Journal ◽

10.1042/bj1330739 ◽

1973 ◽

Vol 133 (4) ◽

pp. 739-747 ◽

Cited By ~ 2

Author(s):

A. Robinson ◽

J. Sykes

Keyword(s):

Escherichia Coli ◽

Protein Interactions ◽

Ribosomal Proteins ◽

Ribosomal Subunit ◽

23S Rrna ◽

Core Particle ◽

Protein Loss ◽

E Coli ◽

Rrna Species ◽

Rhodopseudomonas Spheroides

1. The behaviour of the large ribosomal subunit from Rhodopseudomonas spheroides (45S) has been compared with the 50S ribosome from Escherichia coli M.R.E. 600 (and E. coli M.R.E. 162) during unfolding by removal of Mg2+ and detachment of ribosomal proteins by high univalent cation concentrations. The extent to which these processes are reversible with these ribosomes has also been examined. 2. The R. spheroides 45S ribosome unfolds relatively slowly but then gives rise directly to two ribonucleoprotein particles (16.6S and 13.7S); the former contains the intact primary structure of the 16.25S rRNA species and the latter the 15.00S rRNA species of the original ribosome. No detectable protein loss occurs during unfolding. The E. coli ribosome unfolds via a series of discrete intermediates to a single, unfolded ribonucleoprotein unit (19.1S) containing the 23S rRNA and all the protein of the original ribosome. 3. The two unfolded R. spheroides ribonucleoproteins did not recombine when the original conditions were restored but each simply assumed a more compact configuration. Similar treatments reversed the unfolding of the E. coli 50S ribosomes; replacement of Mg2+ caused the refolding of the initial products of unfolding and in the presence of Ni2+ the completely unfolded species (19.1S) again sedimented at the same rate as the original ribosomes (44S). 4. Ribosomal proteins (25%) were dissociated from R. spheroides 45S ribosomes by dialysis against a solution with a Na+/Mg2+ ratio of 250:1. During this process two core particles were formed (21.2S and 14.2S) and the primary structures of the two original rRNA species were conserved. This dissociation was not reversed. With E. coli 50S approximately 15% of the original ribosomal protein was dissociated, a single 37.6S core particle was formed, the 23S rRNA remained intact and the ribosomal proteins would reassociate with the core particle to give a 50S ribosome. 5. The ribonuclease activities in R. spheroides 45S and E. coli M.R.E. 600 and E. coli M.R.E. 162 50S ribosomes are compared. 6. The observations concerning unfolding and dissociation are consistent with previous reports showing the unusual rRNA complement of the mature R. spheroides 45S ribosome and show the dependence of these events upon the rRNA and the importance of protein–protein interactions in the structure of the R. spheroides ribosome.

Download Full-text

Studies of the Interaction of Escherichia coli YjeQ with the Ribosome In Vitro

Journal of Bacteriology ◽

10.1128/jb.186.5.1381-1387.2004 ◽

2004 ◽

Vol 186 (5) ◽

pp. 1381-1387 ◽

Cited By ~ 75

Author(s):

Denis M. Daigle ◽

Eric D. Brown

Keyword(s):

Escherichia Coli ◽

Ribosomal Subunit ◽

Gtp Hydrolysis ◽

E Coli ◽

Cell Extracts ◽

30S Subunit ◽

Ob Fold ◽

Nucleotide Binding Proteins ◽

Dependent Interaction

ABSTRACT 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.

Download Full-text

Multiple in vivo pathways for Escherichia coli small ribosomal subunit assembly occur on one pre-rRNA

Nature Structural & Molecular Biology ◽

10.1038/nsmb.2887 ◽

2014 ◽

Vol 21 (10) ◽

pp. 937-943 ◽

Cited By ~ 16

Author(s):

Neha Gupta ◽

Gloria M Culver

Keyword(s):

Escherichia Coli ◽

Ribosomal Subunit ◽

Small Ribosomal Subunit ◽

Subunit Assembly

Download Full-text

Two New Point Mutations at A2062 Associated with Resistance to 16-Membered Macrolide Antibiotics in Mutant Strains ofMycoplasma hominis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.10.2958-2960.2001 ◽

2001 ◽

Vol 45 (10) ◽

pp. 2958-2960 ◽

Cited By ~ 26

Author(s):

Pio Maria Furneri ◽

Giancarlo Rappazzo ◽

Maria Pia Musumarra ◽

Patrizia Di Pietro ◽

Lucrezia S. Catania ◽

...

Keyword(s):

Escherichia Coli ◽

Point Mutations ◽

Mycoplasma Hominis ◽

23S Rrna ◽

Macrolide Antibiotics ◽

Rrna Gene ◽

In Vitro Exposure ◽

Mutant Strains ◽

23S Rrna Gene

ABSTRACT We describe two mutants of Mycoplasma hominis PG-21 which show resistance to 16-membered macrolides but susceptibility to lincosamides, obtained by in vitro exposure to increasing doses of josamycin. The 23S rRNA gene showed that each had a mutation (A2062G and A2062T) corresponding to nucleotide 2062 in Escherichia coli, which was associated with the acquired phenotype.

Download Full-text