The Conserved A-site Finger of the 23S rRNA: Just One of the Intersubunit Bridges or a Part of the Allosteric Communication Pathway?

Сергиев, Петр В.; Kiparisov, Sergey V.; Burakovsky, Dmitry E.; Lesnyak, Dmitry V.; Leonov, Andrei; Bøgdanov, A.; Dontsova, Olga А.

doi:10.1016/j.jmb.2005.08.006

Cited by 37 publications

(41 citation statements)

References 31 publications

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“…Although the U860C and G864A mutations are lethal, the disruption of the A site finger by deletion of the tip of H38 (the ⌬B1a mutation) has only a mild phenotype (31,44,45). Furthermore, the U860C mutation was severely deleterious even in the context of the ⌬B1a deletion (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…7) (42, 43). Interestingly, however, deletion of up to 34 nt at the tip of H38 that disrupts the bridge results in only mild phenotypes (31,44,45). To investigate whether the deleterious effect of H38 point mutations we observed in our experiments is related to the function of this rRNA element as an intersubunit bridge, we engineered two new mutants.…”

Section: Selection Of Deleterious Mutations In 23mentioning

confidence: 99%

“…Fragment exchange was used for introduction of the 34-nt deletion in helix 38 in the pLK35 plasmid. The XbaI-SphI segment was excised from the plasmid pLK1192U that carried a deletion of the 872-905 segment of 23 S rRNA (31), purified in 1% SeaPlaque agarose gel (Cambrex), and cloned into pLK35 vector cut with the same restriction enzymes. The U860C mutation was then introduced into the resulting plasmid using the QuikChange XL site-directed mutagenesis kit.…”

Section: Mutagenesis and Construction Of Segment-mutant Libraries-mentioning

confidence: 99%

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Potential New Antibiotic Sites in the Ribosome Revealed by Deleterious Mutations in RNA of the Large Ribosomal Subunit

Yassin¹,

Mankin

2007

Journal of Biological Chemistry

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The ribosome is the main target for antibiotics that inhibit protein biosynthesis. Despite the chemical diversity of the known antibiotics that affect functions of the large ribosomal subunit, these drugs act on only a few sites corresponding to some of the known functional centers. We have used a genetic approach for identifying structurally and functionally critical sites in the ribosome that can be used as new antibiotic targets. By using randomly mutagenized rRNA genes, we mapped rRNA sites where nucleotide alterations impair the ribosome function or assembly and lead to a deleterious phenotype. A total of 77 single-point deleterious mutations were mapped in 23 S rRNA and ranked according to the severity of their deleterious phenotypes. Many of the mutations mapped to familiar functional sites that are targeted by known antibiotics. However, a number of mutations were located in previously unexplored regions. The distribution of the mutations in the spatial structure of the ribosome showed a strong bias, with the strongly deleterious mutations being mainly localized at the interface of the large subunit and the mild ones on the solvent side. Five sites where deleterious mutations tend to cluster within discrete rRNA elements were identified as potential new antibiotic targets. One of the sites, the conserved segment of helix 38, was studied in more detail. Although the ability of the mutant 50 S subunits to associate with 30 S subunits was impaired, the lethal effect of mutations in this rRNA element was unrelated to its function as an intersubunit bridge. Instead, mutations in this region had a profound deleterious effect on the ribosome assembly.

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Section: Discussionmentioning

confidence: 99%

Section: Selection Of Deleterious Mutations In 23mentioning

confidence: 99%

Section: Mutagenesis and Construction Of Segment-mutant Libraries-mentioning

confidence: 99%

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Potential New Antibiotic Sites in the Ribosome Revealed by Deleterious Mutations in RNA of the Large Ribosomal Subunit

Yassin¹,

Mankin

2007

Journal of Biological Chemistry

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“…For translation fidelity assays, the cells were harvested at the same stage of growth, namely, the mid-log phase. Translational fidelity was measured using a set of LacZ reporter strains (pSG or f9CSH) (O'Connor and Dahlberg 1993;Sergiev et al 2005). E. coli strain MC140 carrying one of the reporter plasmids was transformed by one of the plasmids carrying a wild-type or mutant 23S rRNA gene.…”

Section: In Vivo Assaysmentioning

confidence: 99%

Mutations at the accommodation gate of the ribosome impair RF2-dependent translation termination

Burakovsky¹,

Сергиев²,

Steblyanko³

et al. 2010

RNA

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During protein synthesis, aminoacyl-tRNA (aa-tRNA) and release factors 1 and 2 (RF1 and RF2) have to bind at the catalytic center of the ribosome on the 50S subunit where they take part in peptide bond formation or peptidyl-tRNA hydrolysis, respectively. Computer simulations of aa-tRNA movement into the catalytic site (accommodation) suggested that three nucleotides of 23S rRNA, U2492, C2556, and C2573, form a ''gate'' at which aa-tRNA movement into the A site is retarded. Here we examined the role of nucleotides C2573 of 23S rRNA, a part of the putative accommodation gate, and of the neighboring A2572 for aa-tRNA binding followed by peptide bond formation and for the RF2-dependent peptide release. Mutations at the two positions did not affect aa-tRNA accommodation, peptide bond formation, or the fidelity of aa-tRNA selection, but impaired RF2-catalyzed peptide release. The data suggest that the ribosome is a robust machine that allows rapid aa-tRNA accommodation despite the defects at the accommodation gate. In comparison, peptide release by RF2 appears more sensitive to these mutations, due to slower accommodation of the factor or effects on RF2 positioning in the A site.

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“…Previous studies had shown a slight growth defect from truncating helix 38 and disrupting B1a (16,19,31). We measured growth rates for SQ171 cells expressing either wild-type or C889U mutant rRNA and found them to be identical (with a 59-min doubling time).…”

Section: Resultsmentioning

confidence: 88%

rRNA Mutations That Inhibit Transfer-Messenger RNA Activity on Stalled Ribosomes

et al. 2010

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In eubacteria, stalled ribosomes are rescued by a conserved quality-control mechanism involving transfermessenger RNA (tmRNA) and its protein partner, SmpB. Mimicking a tRNA, tmRNA enters stalled ribosomes, adds Ala to the nascent polypeptide, and serves as a template to encode a short peptide that tags the nascent protein for destruction. To further characterize the tagging process, we developed two genetic selections that link tmRNA activity to cell death. These negative selections can be used to identify inhibitors of tagging or to identify mutations in key residues essential for ribosome rescue. Little is known about which ribosomal elements are specifically required for tmRNA activity. Using these selections, we isolated rRNA mutations that block the rescue of ribosomes stalled at rare Arg codons or at the inefficient termination signal Pro-opal. We found that deletion of A1150 in the 16S rRNA blocked tagging regardless of the stalling sequence, suggesting that it inhibits tmRNA activity directly. The C889U mutation in 23S rRNA, however, lowered tagging levels at Pro-opal and rare Arg codons, but not at the 3 end of an mRNA lacking a stop codon. We concluded that the C889U mutation does not inhibit tmRNA activity per se but interferes with an upstream step intermediate between stalling and tagging. C889 is found in the A-site finger, where it interacts with the S13 protein in the small subunit (forming intersubunit bridge B1a).

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The Conserved A-site Finger of the 23S rRNA: Just One of the Intersubunit Bridges or a Part of the Allosteric Communication Pathway?

Cited by 37 publications

References 31 publications

Potential New Antibiotic Sites in the Ribosome Revealed by Deleterious Mutations in RNA of the Large Ribosomal Subunit

Potential New Antibiotic Sites in the Ribosome Revealed by Deleterious Mutations in RNA of the Large Ribosomal Subunit

Mutations at the accommodation gate of the ribosome impair RF2-dependent translation termination

rRNA Mutations That Inhibit Transfer-Messenger RNA Activity on Stalled Ribosomes

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