Genetic evidence against the 16S ribosomal RNA helix 27 conformational switch model

Rodriguez-Correa, Daniel; Dahĺberg, Albert E.

doi:10.1261/rna.5172104

Cited by 24 publications

(21 citation statements)

References 38 publications

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“…The effects of mutations of A1191 observed here may offer an explanation for the earlier observations of synergistic effects of mutations in h27 and in the pSTL102 plasmid, which has two mutations, C1192U in 16 S rRNA and A2058G in 23 S rRNA (39). In fact, if replacements of C1192 had similar effects as those of the adjacent A1191, then an effect on the conformation of h27 would be expected.…”

Section: Effects Of Mutations On 30 S Subunit Structure and Associatisupporting

confidence: 65%

Involvement of Helix 34 of 16 S rRNA in Decoding and Translocation on the Ribosome

Kubarenko

Сергиев

Wintermeyer

et al. 2006

Journal of Biological Chemistry

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Helix 34 of 16 S rRNA is located in the head of the 30 S ribosomal subunit close to the decoding center and has been invoked in a number of ribosome functions. In the present work, we have studied the effects of mutations in helix 34 both in vivo and in vitro. Several nucleotides in helix 34 that are either highly conserved or form important tertiary contacts in 16 S rRNA (U961, C1109, A1191, and A1201) were mutated, and the mutant ribosomes were expressed in the Escherichia coli MC250 ⌬7 strain that lacks all seven chromosomal rRNA operons. Mutations at positions A1191 and U961 reduced the efficiency of subunit association and resulted in structural rearrangements in helix 27 (position 908) and helix 31 (position 974) of 16 S rRNA. All mutants exhibited increased levels of frameshifting and nonsense readthrough. The effects on frameshifting were specific in that ؊1 frameshifting was enhanced with mutant A1191G and ؉1 frameshifting with the other mutants. Mutations of A1191 moderately (ϳ2-fold) inhibited tRNA translocation. No significant effects were found on efficiency and rate of initiation, misreading of sense codons, or binding of tRNA to the E site. The data indicate that helix 34 is involved in controlling the maintenance of the reading frame and in tRNA translocation.

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Section: Effects Of Mutations On 30 S Subunit Structure and Associatisupporting

confidence: 65%

Involvement of Helix 34 of 16 S rRNA in Decoding and Translocation on the Ribosome

Kubarenko

Сергиев

Wintermeyer

et al. 2006

Journal of Biological Chemistry

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“…However, a recent report indicated that they may exhibit synthetic lethality when combined with specific other mutations (37). Although we have not tested all of the selected mutations in segregation from the resident pLK45 mutations, 10 of the individual deleterious mutations tested in the specialized ribosome system showed severe defects in translation, and several other mutations from our collection were previously individually engineered in 16S rRNA and shown to inhibit cell growth (Table 1).…”

Section: Discussionmentioning

confidence: 98%

Deleterious mutations in small subunit ribosomal RNA identify functional sites and potential targets for antibiotics

Yassin

Fredrick

Mankin

2005

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

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Many clinically useful antibiotics interfere with protein synthesis in bacterial pathogens by inhibiting ribosome function. The sites of action of known drugs are limited in number, are composed primarily of ribosomal RNA (rRNA), and coincide with functionally critical centers of the ribosome. Nucleotide alterations within such sites are often deleterious. To identify functional sites and potential sites of antibiotic action in the ribosome, we prepared a random mutant library of rRNA genes and selected dominant mutations in 16S rRNA that interfere with cell growth. Fifty-three 16S rRNA positions were identified whose mutation inhibits protein synthesis. Mutations were ranked according to the severity of the phenotype, and the detrimental effect of several mutations on translation was verified in a specialized ribosome system. Analysis of the polysome profiles of mutants suggests that the majority of the mutations directly interfered with ribosome function, whereas a smaller fraction of mutations affected assembly of the small ribosomal subunit. Twelve of the identified mutations mapped to sites targeted by known antibiotics, confirming that deleterious mutations can be used to identify antibiotic targets. About half of the mutations coincided with known functional sites in the ribosome, whereas the rest of the mutations affected ribosomal sites with less clear functional significance. Four clusters of deleterious mutations in otherwise unremarkable ribosomal sites were identified, suggesting their functional importance and potential as antibiotic targets.16S rRNA ͉ 30S subunit ͉ resistance ͉ ribosome ͉ translation W ith a molecular mass of Ϸ2.5 million Da and Ͼ50 different RNA and protein building blocks, the ribosome represents one of the largest and most complex enzymes in the cell. Ribosomal RNA (rRNA) accounts for two-thirds of the ribosome and is responsible for its main functions in protein synthesis: interpretation of genetic information and polymerization of amino acids into a polypeptide. rRNA is also intimately involved in known auxiliary activities of the ribosome, such as nascent peptide release, binding of translation factors, GTP hydrolysis, etc. (reviewed in ref. 1).The ribosome is the predominant antibiotic target in the bacterial cell. A large variety of natural and synthetic antibiotics interfere with translation by binding to rRNA and preventing the correct placement of ribosomal ligands, corrupting rRNA structure, or affecting conformational flexibility of rRNA (2). Advantages of the ribosome as an antibiotic target may stem from its RNA-based design. The multiplicity of rRNA genes in microbial genomes makes it difficult for a microorganism to develop resistance by mutating the drug-binding site (3). Furthermore, RNA offers fewer mutational options than protein enzymes (3 versus 19, respectively), which makes it more difficult for a microbial pathogen to ''find'' a mutation that would reduce antibiotic binding without compromising functional integrity of the enzyme. In the clinical setting, ...

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“…Subsequent crystal structures of ''open'' and ''closed'' forms of the 30S subunit in complex with aminoglycoside antibiotics consistently depict H27 in the 885 conformation (Ogle et al 2002), as do crystal structures of hyperaccurate ribosomes containing S12 mutations (Vila-Sanjurjo et al 2003). Follow-up studies by the Dahlberg group revealed that their previous findings originated from a synergistic effect between H27 and selective marker mutations (Rodriguez-Correa and Dahlberg 2004), suggesting that H27 does not need to switch between conformations during translation. In contrast, an isolated H27 exists in a rapid dynamic equilibrium between the 885 and 888 conformations, indicative of a low energy barrier for conformational switching (Hoerter et al 2004).…”

Section: Introductionmentioning

confidence: 92%

Solution probing of metal ion binding by helix 27 fromEscherichia coli16S rRNA

Lambert

Hoerter

Pereira

et al. 2005

RNA

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Helix (H)27 from Escherichia coli 16S ribosomal (r)RNA is centrally located within the small (30S) ribosomal subunit, immediately adjacent to the decoding center. Bacterial 30S subunit crystal structures depicting Mg 2+ binding sites resolve two magnesium ions within the vicinity of H27: one in the major groove of the G886-U911 wobble pair, and one within the GCAA tetraloop. Binding of such metal cations is generally thought to be crucial for RNA folding and function. To ask how metal ion-RNA interactions in crystals compare with those in solution, we have characterized, using solution NMR spectroscopy, Tb 3+ footprinting and time-resolved fluorescence resonance energy transfer (tr-FRET), location, and modes of metal ion binding in an isolated H27. NMR and Tb 3+ footprinting data indicate that solution secondary structure and Mg 2+ binding are generally consistent with the ribosomal crystal structures. However, our analyses also suggest that H27 is dynamic in solution and that metal ions localize within the narrow major groove formed by the juxtaposition of the loop E motif with the tandem G894-U905 and G895-U904 wobble pairs. In addition, tr-FRET studies provide evidence that Mg 2+ uptake by the H27 construct results in a global lengthening of the helix. We propose that only a subset of H27-metal ion interactions has been captured in the crystal structures of the 30S ribosomal subunit, and that small-scale structural dynamics afforded by solution conditions may contribute to these differences. Our studies thus highlight an example for differences between RNA-metal ion interactions observed in solution and in crystals.

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Genetic evidence against the 16S ribosomal RNA helix 27 conformational switch model

Cited by 24 publications

References 38 publications

Involvement of Helix 34 of 16 S rRNA in Decoding and Translocation on the Ribosome

Involvement of Helix 34 of 16 S rRNA in Decoding and Translocation on the Ribosome

Deleterious mutations in small subunit ribosomal RNA identify functional sites and potential targets for antibiotics

Solution probing of metal ion binding by helix 27 fromEscherichia coli16S rRNA

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