The first determination of pseudouridine residues in 23S ribosomal RNA from hyperthermophilic <i>Archaea Sulfolobus acidocaldarius</i>

SummaryPseudouridine (5-ribosyluracil ) is a ubiquitous yet enigmatic constituent of structural RNAs (transfer, ribosomal, small nuclear, and small nucleolar). Although pseudouridine (W ) was the rst modi ed nucleoside to be discovered in RNA, and is the most abundant, its biosynthesis and biological roles have remained poorly understood since its identi cation as a " fth nucleoside" in RNA. Recently, a combination of biochemical, biophysical, and genetic approaches has helped to illuminate the structural consequences of W in polyribonucleotides, the biochemical mechanism of U! W

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“…Notably, the low W content in archaebacterial LSU rRNA (21,28) is more like that seen in eubacteria than in eukaryotes.…”

Section: Ribosomal Rnasmentioning

confidence: 88%

Pseudouridine in RNA: What, Where, How, and Why

2000

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“…The number and locations of these modifications vary between organisms; E. coli, Bacillus subtilis, and Deinococcus radiodurans have ⌿ at positions 1911, 1915, and 1917, and ⌿1915 is methylated in at least E. coli and D. radiodurans. Thermus thermophilus has ⌿ at positions 1911 and 1917, Haloarcula marismortui has ⌿ at 1915 and 1917, while Sulfolobus acidocaldarius has a single ⌿ at position 1917 (7,20,22,28). In Saccharomyces cerevisiae cytoplasmic ribosomes, ⌿s are found at positions 1915 and 1917, as well as in the stem region of h69 at positions 1921 and 1923 (E. coli numbering) (18).…”

Section: Discussionmentioning

confidence: 99%

Inactivation of the RluD Pseudouridine Synthase Has Minimal Effects on Growth and Ribosome Function in Wild-TypeEscherichia coliandSalmonella enterica

O’Connor

Gregory

2011

J Bacteriol

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The Escherichia coli rluD gene encodes a pseudouridine synthase responsible for the pseudouridine (⌿) modifications at positions 1911, 1915, and 1917 in helix 69 of 23S rRNA. It has been reported that deletion of rluD in K-12 strains of E. coli is associated with extremely slow growth, increased readthrough of stop codons, and defects in 50S ribosomal subunit assembly and 30S-50S subunit association. Suppressor mutations in the prfB and prfC genes encoding release factor 2 (RF2) and RF3 that restore the wild type-growth rate and also correct the ribosomal defects have now been isolated. These suppressors link helix 69 ⌿ residues with the termination phase of protein synthesis. However, further genetic analysis reported here also reveals that the slow growth and other defects associated with inactivation of rluD in E. coli K-12 strains are due to a defective RF2 protein, with a threonine at position 246, which is present in all K-12 strains. This is in contrast to the more typical alanine found at this position in most bacterial RF2s, including those of other E. coli strains. Inactivation of rluD in E. coli strains containing the prfB allele from E. coli B or in Salmonella enterica, both carrying an RF2 with Ala246, has negligible effects on growth, termination, or ribosome function. The results indicate that, in contrast to those in wild bacteria, termination functions in E. coli K-12 strains carrying a partially defective RF2 protein are especially susceptible to perturbation of ribosome-RF interactions, such as that caused by loss of h69 ⌿ modifications.Pseudouridine (⌿) is among the most common posttranscriptional modifications occurring in rRNA and has been found at conserved regions of the rRNAs in organisms from all three kingdoms (28,29). In Escherichia coli, there are 11 ⌿ residues in 16S and 23S rRNAs, and the 7 synthases responsible for these modifications have now been identified (30). The functions of specific ⌿ residues have been investigated by deletion of the corresponding ⌿ synthase genes (rsuA, rluA, rluB, rluC, rluD, rluE, and rluF) and characterization of the resulting deletion mutants. With the notable exception of rluD, deletion of individual ⌿ synthase genes has little effect on cell growth (29). In contrast, inactivation or deletion of rluD has been reported to have profound effects on cell growth, 50S subunit assembly, and subunit association (14, 32). RluD is responsible for ⌿1911, ⌿1915, and ⌿1917 modifications in helix 69 (h69) of 23S rRNA. This functionally important stemloop structure forms a bridge between the 30S and 50S ribosomal subunits and interacts with translation factors and tRNAs at multiple stages of protein synthesis (39).The location of ⌿ residues in h69 and the phenotypes of the ⌬rluD mutants lacking h69 ⌿ residues suggest that these posttranscriptional modifications are important for ribosome function. Strains lacking RluD are extremely unstable and rapidly give rise to faster-growing, suppressor-containing derivatives (12,14). Some insights into h69 ⌿ functions were...

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“…We used the N -cyclohexyl- N ′-(2-morpholinoethyl)-carbodiimide metho- p -toluolsulfonate (CMCT) modification protocol of (42), adapted by (43). CMCT modifications were performed with 0.5 μg of in vitro transcribed archaeal or yeast tRNA Asp .…”

Section: Methodsmentioning

confidence: 99%

Identification of determinants in the protein partners aCBF5 and aNOP10 necessary for the tRNA: 55-synthase and RNA-guided RNA: -synthase activities

Müller

Fourmann

Loegler

et al. 2007

Nucleic Acids Research

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Protein aNOP10 has an essential scaffolding function in H/ACA sRNPs and its interaction with the pseudouridine(Ψ)-synthase aCBF5 is required for the RNA-guided RNA:Ψ-synthase activity. Recently, aCBF5 was shown to catalyze the isomerization of U55 in tRNAs without the help of a guide sRNA. Here we show that the stable anchoring of aCBF5 to tRNAs relies on its PUA domain and the tRNA CCA sequence. Nonetheless, interaction of aNOP10 with aCBF5 can counterbalance the absence of the PUA domain or the CCA sequence and more generally helps the aCBF5 tRNA:Ψ55-synthase activity. Whereas substitution of the aNOP10 residue Y14 by an alanine disturbs this activity, it only impairs mildly the RNA-guided activity. The opposite effect was observed for the aNOP10 variant H31A. Substitution K53A or R202A in aCBF5 impairs both the tRNA:Ψ55-synthase and the RNA-guided RNA:Ψ-synthase activities. Remarkably, the presence of aNOP10 compensates for the negative effect of these substitutions on the tRNA: Ψ55-synthase activity. Substitution of the aCBF5 conserved residue H77 that is expected to extrude the targeted U residue in tRNA strongly affects the efficiency of U55 modification but has no major effect on the RNA-guided activity. This negative effect can also be compensated by the presence of aNOP10.

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The first determination of pseudouridine residues in 23S ribosomal RNA from hyperthermophilic Archaea Sulfolobus acidocaldarius

Abstract: We describe the first identification of pseudouridine (8 8) residues in ribosomal RNA (23S rRNA) of an hyperthermophilic Archaea Sulfolobus acidocaldarius. In contrast to Eucarya rRNA, only six 8 8 residues were detected, which is rather close to the situation in Bacteria.

Cited by 18 publications

References 56 publications

Pseudouridine in RNA: What, Where, How, and Why

Pseudouridine in RNA: What, Where, How, and Why

Inactivation of the RluD Pseudouridine Synthase Has Minimal Effects on Growth and Ribosome Function in Wild-TypeEscherichia coliandSalmonella enterica

Identification of determinants in the protein partners aCBF5 and aNOP10 necessary for the tRNA: 55-synthase and RNA-guided RNA: -synthase activities

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