Genetic and structural analysis of base substitutions in the central pseudoknot of <i>Thermus thermophilus</i> 16S ribosomal RNA

Gregory, Steven T.; Dahĺberg, Albert E.

doi:10.1261/rna.1374809

Cited by 19 publications

(26 citation statements)

References 44 publications

(57 reference statements)

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“…T. thermophilus ribosomes were used because of their ability to produce crystals that are amenable for X-ray structure determination (25). One of the two 16S rRNA genes (rrsB) was first replaced with the mutant allele, and then the other (rrsA) was replaced with the null allele ΔrrsA::htk1, marked with the high-temperature kanamycin resistance 1 gene (26). In both Escherichia coli (strain Δ7 prrn) and T. thermophilus, G299A conferred a larger growth defect than G347U.…”

Section: Resultsmentioning

confidence: 99%

“…T. thermophilus strains expressing homogeneous populations of mutant ribosomes were constructed as follows. A ∼1,500-bp DNA fragment that includes the 5′ two-thirds of rrsB and adjacent DNA upstream was amplified from the T. thermophilus genome and cloned into pUC18-htk1, a vector encoding a thermostable kanamycin adenyltransferase (26,43). The resulting plasmid pMD3 was subjected to sitedirected mutagenesis to produce the derivatives pMD5 and pMD6, with mutations corresponding to 16S rRNA substitutions G347U and G299A, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Reorganization of an intersubunit bridge induced by disparate 16S ribosomal ambiguity mutations mimics an EF-Tu-bound state

Fagan

Dunkle

Maehigashi

et al. 2013

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

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After four decades of research aimed at understanding tRNA selection on the ribosome, the mechanism by which ribosomal ambiguity (ram) mutations promote miscoding remains unclear. Here, we present two X-ray crystal structures of the Thermus thermophilus 70S ribosome containing 16S rRNA ram mutations, G347U and G299A. Each of these mutations causes miscoding in vivo and stimulates elongation factor thermo unstable (EF-Tu)-dependent GTP hydrolysis in vitro. Mutation G299A is located near the interface of ribosomal proteins S4 and S5 on the solvent side of the subunit, whereas G347U is located 77 Å distant, at intersubunit bridge B8, close to where EF-Tu engages the ribosome. Despite these disparate locations, both mutations induce almost identical structural rearrangements that disrupt the B8 bridge-namely, the interaction of h8/h14 with L14 and L19. This conformation most closely resembles that seen upon EF-Tu·GTP·aminoacyl-tRNA binding to the 70S ribosome. These data provide evidence that disruption and/or distortion of B8 is an important aspect of GTPase activation. We propose that, by destabilizing B8, G299A and G347U reduce the energetic cost of attaining the GTPase-activated state and thereby decrease the stringency of decoding. This previously unappreciated role for B8 in controlling the decoding process may hold relevance for many other ribosomal mutations known to influence translational fidelity.T he molecular mechanisms controlling the fidelity of DNA replication, transcription, and translation have been areas of intense interest since the discovery of the genetic code. Thermodynamic differences between standard Watson-Crick and alternative (e.g., wobble) base pairs in solution are insufficient to explain the high fidelity for any of the three polymerase reactions of the central dogma (1), indicating an active role for the enzymes in substrate selectivity (1-3). Mechanistic studies of polymerases have revealed some common themes, such as the specific recognition of Watson-Crick base pair geometry, larger forward rate constants for correct substrates (induced fit), separate opportunities for incorrect substrate rejection (kinetic proofreading), and postincorporation correction mechanisms (1-5).During translation, the ribosome must select aminoacyl-tRNA (aa-tRNA) substrates based on the mRNA sequence. Extensive biochemical studies have shed light on the kinetics of this decoding process (reviewed in ref. 6). The aa-tRNA is delivered to the ribosome as part of a ternary complex (TC) with elongation factor thermo unstable (EF-Tu) and GTP. Initial binding of TC, mediated primarily by L7/L12 of the 50S subunit, is followed by the sampling of codon-anticodon interactions in the 30S A site. Codon-anticodon pairing leads to GTPase activation and GTP hydrolysis, which allows release of the acceptor end of aa-tRNA from EF-Tu. The aa-tRNA then either moves completely into the ribosomal A site (a step termed accommodation), where it can participate in peptide bond formation, or is rejected and released into solution...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Reorganization of an intersubunit bridge induced by disparate 16S ribosomal ambiguity mutations mimics an EF-Tu-bound state

Fagan

Dunkle

Maehigashi

et al. 2013

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

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“…As the replication origin of pUC-derived plasmids is not active in T. thermophilus, transformation with the knockout constructs to Kan r results from homologous recombination between the UHR and DHR with their respective chromosomal sequences. In contrast to findings with E. coli (38,39), double crossovers work very efficiently in T. thermophilus, without the need to select for single insertions and subsequent cointegrate resolution (17,35,40). While the latter approach does have the advantage of allowing the use of a single plasmid construct for insertion of pheS and its replacement, our preference is to construct an intermediate double-crossover strain as it allows replacement by transformation with genomic DNA as well as plasmids.…”

Section: Resultsmentioning

confidence: 99%

“…The T. thermophilus HB27 genome produces 16S rRNA from two loci (rrsA and rrsB) that are unlinked to two rRNA operons, each encoding 23S rRNA (rrlA or rrlB), 5S rRNA (rrfA), and tRNA GCC Gly (glyT) as first described by Hartmann and Erdmann (41) and later indicated by the completed genome sequence (5). Our previous studies have shown that T. thermophilus is capable of surviving on a single rrs gene (35). Such deletion strains facilitate the genetic analysis of rRNA genes and allow the isolation of mutants with recessive antibiotic resistance mutations (35).…”

Section: Resultsmentioning

confidence: 99%

“…Our previous studies have shown that T. thermophilus is capable of surviving on a single rrs gene (35). Such deletion strains facilitate the genetic analysis of rRNA genes and allow the isolation of mutants with recessive antibiotic resistance mutations (35). Since two of the available selectable markers confer resistance to the ribosome-targeted antibiotics kanamycin and hygromycin B, having a ⌬rrsB strain lacking such drug-resistance genes would be of some advantage for investigating mechanisms of antibiotic resistance due to mutations in the ribosome.…”

Section: Resultsmentioning

confidence: 99%

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Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker

et al. 2015

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Thermus thermophilus is an extremely thermophilic bacterium that is widely used as a model thermophile, in large part due to its amenability to genetic manipulation. Here we describe a system for the introduction of genomic point mutations or deletions using a counterselectable marker consisting of a conditionally lethal mutant allele of pheS encoding the phenylalanyl-tRNA synthetase ␣-subunit. Mutant PheS with an A294G amino acid substitution renders cells sensitive to the phenylalanine analog pchlorophenylalanine. Insertion of the mutant pheS allele via a linked kanamycin resistance gene into a chromosomal locus provides a gene replacement intermediate that can be removed by homologous recombination using p-chlorophenylalanine as a counterselective agent. This selection is suitable for the sequential introduction of multiple mutations to produce a final strain unmarked by an antibiotic resistance gene. We demonstrated the utility of this method by constructing strains bearing either a point mutation in or a precise deletion of the rrsB gene encoding 16S rRNA. We also used this selection to identify spontaneous, large-scale deletions in the pTT27 megaplasmid, apparently mediated by either of the T. thermophilus insertion elements ISTth7 and ISTth8. One such deletion removed 121 kb, including 118 genes, or over half of pTT27, including multiple sugar hydrolase genes, and facilitated the development of a plasmid-encoded reporter system based on ␤-galactosidase. The ability to introduce mutations ranging from single base substitutions to large-scale deletions provides a potentially powerful tool for engineering the genome of T. thermophilus and possibly other thermophiles as well. IMPORTANCEThermus thermophilus is an extreme thermophile that has played an important part in the development of both biotechnology and basic biological research. Its suitability as a genetic model system is established by its natural competence for transformation, but the scarcity of genetic tools limits the kinds of manipulations that can currently be performed. We have developed a counterselectable marker that allows the introduction of unmarked deletions and point mutations into the T. thermophilus genome. We find that this marker can also be used to select large chromosomal deletions apparently resulting from aberrant transposition of endogenous insertion sequences. This system has the potential to advance the genetic manipulation of this important model organism.

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Environmental Reservoirs of Resistance Genes in Antibiotic‐Producing Bacteria and Their Possible Impact on the Evolution of Antibiotic Resistance

Laskaris

Gaze

Wellington

2011

Antimicrobial Resistance in the Environment

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Genetic and structural analysis of base substitutions in the central pseudoknot of Thermus thermophilus 16S ribosomal RNA

Cited by 19 publications

References 44 publications

Reorganization of an intersubunit bridge induced by disparate 16S ribosomal ambiguity mutations mimics an EF-Tu-bound state

Reorganization of an intersubunit bridge induced by disparate 16S ribosomal ambiguity mutations mimics an EF-Tu-bound state

Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker

Environmental Reservoirs of Resistance Genes in Antibiotic‐Producing Bacteria and Their Possible Impact on the Evolution of Antibiotic Resistance

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