Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Siibak, Triinu; Rèmme, Jaanus

doi:10.1261/rna.2160010

Cited by 57 publications

(84 citation statements)

References 46 publications

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“…Nascent 23S rRNA seems to be the natural substrate for YhiR. This result is in agreement with an earlier study (Siibak and Remme 2010) indicating that modification of the 23S rRNA nucleotide A2030 occurs early in the 50S subunit assembly.…”

Section: Yhir Methylates 23s Rrna In Vitrosupporting

confidence: 93%

The last rRNA methyltransferase of E. coli revealed: The yhiR gene encodes adenine-N6 methyltransferase specific for modification of A2030 of 23S ribosomal RNA

Golovina

Dzama

Osterman

et al. 2012

RNA

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The ribosomal RNA (rRNA) of Escherichia coli contains 24 methylated residues. A set of 22 methyltransferases responsible for modification of 23 residues has been described previously. Herein we report the identification of the yhiR gene as encoding the enzyme that modifies the 23S rRNA nucleotide A2030, the last methylated rRNA nucleotide whose modification enzyme was not known. YhiR prefers protein-free 23S rRNA to ribonucleoprotein particles containing only part of the 50S subunit proteins and does not methylate the assembled 50S subunit. We suggest renaming the yhiR gene to rlmJ according to the rRNA methyltransferase nomenclature. The phenotype of yhiR knockout gene is very mild under various growth conditions and at the stationary phase, except for a small growth advantage at anaerobic conditions. Only minor changes in the total E. coli proteome could be observed in a cell devoid of the 23S rRNA nucleotide A2030 methylation.

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Section: Yhir Methylates 23s Rrna In Vitrosupporting

confidence: 93%

The last rRNA methyltransferase of E. coli revealed: The yhiR gene encodes adenine-N6 methyltransferase specific for modification of A2030 of 23S ribosomal RNA

Golovina

Dzama

Osterman

et al. 2012

RNA

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“…Given that most 50S subunits in cells are associated with 30S subunits at sufficient concentrations of Mg 2+ , it is reasonable to speculate that the physiological substrate for RlmE is not the 50S subunit, but rather the 45S precursor, because Um2552 in H92 of 23S rRNA is located on the interface side of the 50S subunit (24)(25)(26). Quantitative analyses of rRNA modifications by HPLC/MS have shown that the level of Um2552 surges during late steps of 50S assembly (32,33), and these results are consistent with our observations. To obtain insights into the function of an RNA modification responsible for a specific biological process, it is necessary to determine whether the RNA-modifying enzyme or the RNA modification itself plays a pivotal role in the process.…”

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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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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“…5). RluD is, according to current knowledge, the only pseudouridine synthase acting during late stages of ribosome assembly (Siibak and Remme 2010).…”

Section: Discussionmentioning

confidence: 99%

Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD

Ero¹,

Leppik²,

Liiv³

et al. 2010

RNA

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Along the ribosome assembly pathway, various ribosomal RNA processing and modification reactions take place. Stem-loop 69 in the large subunit of Escherichia coli ribosomes plays a substantial role in ribosome functioning. It contains three highly conserved pseudouridines synthesized by pseudouridine synthase RluD. One of the pseudouridines is further methylated by RlmH. In this paper we show that RlmH has unique substrate specificity among rRNA modification enzymes. It preferentially methylates pseudouridine and less efficiently uridine. Furthermore, RlmH is the only known modification enzyme that is specific to 70S ribosomes. Kinetic parameters determined for RlmH are the following: The apparent K M for substrate 70S ribosomes is 0.51 6 0.06 mM, and for cofactor S-adenosyl-L-methionine 27 6 3 mM; the k cat values are 4.95 6 1.10 min À1 and 6.4 6 1.3 min À1, respectively. Knowledge of the substrate specificity and the kinetic parameters of RlmH made it possible to determine the kinetic parameters for RluD as well. The K M value for substrate 50S subunits is 0.98 6 0.18 mM and the k cat value is 1.97 6 0.46 min À1. RluD is the first rRNA pseudouridine synthase to be kinetically characterized. The determined rates of RluD-and RlmH-directed modifications of 23S rRNA are compatible with the rate of 50S assembly in vivo. The fact that RlmH requires 30S subunits demonstrates the dependence of 50S subunit maturation on the simultaneous presence of 30S subunits.

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Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Cited by 57 publications

References 46 publications

The last rRNA methyltransferase of E. coli revealed: The yhiR gene encodes adenine-N6 methyltransferase specific for modification of A2030 of 23S ribosomal RNA

The last rRNA methyltransferase of E. coli revealed: The yhiR gene encodes adenine-N6 methyltransferase specific for modification of A2030 of 23S ribosomal RNA

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

Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD

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