RESUMEN
The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin.
Asunto(s)
Antibacterianos , Farmacorresistencia Bacteriana , Antibacterianos/farmacología , Antibacterianos/química , Farmacorresistencia Bacteriana/efectos de los fármacos , Ribosomas/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , ARN Ribosómico 23S/química , ARN Ribosómico 23S/metabolismo , Metiltransferasas/metabolismo , Metiltransferasas/química , Metiltransferasas/antagonistas & inhibidores , Metilación , Modelos Moleculares , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Escherichia coli/genéticaRESUMEN
Although ribosomes are generally examined in aggregate, ribosomes can be heterogenous in composition. Evidence is accumulating that changes in ribosome composition may result in altered function, such that ribosome heterogeneity may provide a mechanism to regulate protein synthesis. Ribosome heterogeneity in the human pathogen Francisella tularensis results from incorporation of one of three homologs of bS21, a small ribosomal subunit protein demonstrated to regulate protein synthesis in other bacteria. Loss of one homolog, bS21-2, results in genome-wide post-transcriptional changes in protein abundance. This suggests that bS21-2 can, either directly or indirectly, lead to preferential translation of particular mRNAs. Here, we examine the potential of bS21-2 to function in a leader sequence-dependent manner and to function indirectly, via Hfq. We found that the 5´ untranslated region (UTR) of some bS21-2-responsive genes, including key virulence genes, is sufficient to alter translation in cells lacking bS21-2. We further identify features of a 5´ UTR that allow responsiveness to bS21-2. These include an imperfect Shine-Dalgarno sequence and a particular six nucleotide sequence. Our results are consistent with a model in which a bS21 homolog increases the efficiency of translation initiation through interactions with specific leader sequences. With respect to bS21-2 indirectly regulating translation via the RNA-binding protein Hfq, we found that Hfq controls transcript abundance rather than protein synthesis, impacting virulence gene expression via a distinct mechanism. Together, we determined that ribosome composition in F. tularensis regulates translation in a leader sequence-dependent manner, a regulatory mechanism which may be used in other bacteria. IMPORTANCE Ribosome heterogeneity is common in bacteria, and there is mounting evidence that ribosome composition plays a regulatory role in protein synthesis. However, mechanisms of ribosome-driven gene regulation are not well understood. In the human pathogen Francisella tularensis, which encodes multiple homologs for the ribosomal protein bS21, loss of one homolog impacts protein synthesis and virulence. Here, we explore the mechanism behind bS21-mediated changes in protein synthesis, finding that they can be linked to altered translation initiation and are dependent on specific sequences in the leaders of transcripts. Our data support a model in which ribosome composition regulates gene expression through translation, a strategy that may be conserved in diverse organisms with various sources of ribosome heterogeneity.
Asunto(s)
Francisella tularensis , Humanos , Francisella tularensis/genética , Ribosomas/genética , Proteínas Ribosómicas/genética , Regiones no Traducidas 5' , ARN Mensajero/genéticaRESUMEN
Many antibiotics inhibit bacterial growth by binding to the ribosome and interfering with protein biosynthesis. Macrolides represent one of the most successful classes of ribosome-targeting antibiotics. The main clinically relevant mechanism of resistance to macrolides is dimethylation of the 23S rRNA nucleotide A2058, located in the drug-binding site, a reaction catalyzed by Erm-type rRNA methyltransferases. Here, we present the crystal structure of the Erm-dimethylated 70S ribosome at 2.4 Å resolution, together with the structures of unmethylated 70S ribosome functional complexes alone or in combination with macrolides. Altogether, our structural data do not support previous models and, instead, suggest a principally new explanation of how A2058 dimethylation confers resistance to macrolides. Moreover, high-resolution structures of two macrolide antibiotics bound to the unmodified ribosome reveal a previously unknown role of the desosamine moiety in drug binding, laying a foundation for the rational knowledge-based design of macrolides that can overcome Erm-mediated resistance.
Asunto(s)
Macrólidos/metabolismo , ARN Ribosómico/ultraestructura , Ribosomas/ultraestructura , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Macrólidos/farmacología , Metilación , ARN Ribosómico/genética , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/metabolismo , ARN Ribosómico 23S/ultraestructura , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Ribosomal protein uS5 is an essential component of the small ribosomal subunit that is involved in subunit assembly, maintenance of translational fidelity, and the ribosome's response to the antibiotic spectinomycin. While many of the characterized uS5 mutations that affect decoding map to its interface with uS4, more recent work has shown that residues distant from the uS4-uS5 interface can also affect the decoding process. We targeted one such interface-remote area, the loop 2 region (residues 20 to 31), for mutagenesis in Escherichia. coli and generated 21 unique mutants. A majority of the loop 2 alterations confer resistance to spectinomycin and affect the fidelity of translation. However, only a minority show altered rRNA processing or ribosome biogenesis defects.
Asunto(s)
Escherichia coli/efectos de los fármacos , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Ribosomas/efectos de los fármacos , Espectinomicina/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutación , Biosíntesis de Proteínas , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Ribosomal proteins S4 and S5 participate in the decoding and assembly processes on the ribosome and the interaction with specific antibiotic inhibitors of translation. Many of the characterized mutations affecting these proteins decrease the accuracy of translation, leading to a ribosomal-ambiguity phenotype. Structural analyses of ribosomal complexes indicate that the tRNA selection pathway involves a transition between the closed and open conformations of the 30S ribosomal subunit and requires disruption of the interface between the S4 and S5 proteins. In agreement with this observation, several of the mutations that promote miscoding alter residues located at the S4-S5 interface. Here, the Escherichia coli rpsD and rpsE genes encoding the S4 and S5 proteins were targeted for mutagenesis and screened for accuracy-altering mutations. While a majority of the 38 mutant proteins recovered decrease the accuracy of translation, error-restrictive mutations were also recovered; only a minority of the mutant proteins affected rRNA processing, ribosome assembly, or interactions with antibiotics. Several of the mutations affect residues at the S4-S5 interface. These include five nonsense mutations that generate C-terminal truncations of S4. These truncations are predicted to destabilize the S4-S5 interface and, consistent with the domain closure model, all have ribosomal-ambiguity phenotypes. A substantial number of the mutations alter distant locations and conceivably affect tRNA selection through indirect effects on the S4-S5 interface or by altering interactions with adjacent ribosomal proteins and 16S rRNA.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Ribosómicas/metabolismo , Bacteriocinas , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Mutagénesis , Péptidos , Proteínas Ribosómicas/genéticaRESUMEN
UNLABELLED: The bacterial ribosome and its associated translation factors are frequent targets of antibiotics, and antibiotic resistance mutations have been found in a number of these components. Such mutations can potentially interact with one another in unpredictable ways, including the phenotypic suppression of one mutation by another. These phenotypic interactions can provide evidence of long-range functional interactions throughout the ribosome and its functional complexes and potentially give insights into antibiotic resistance mechanisms. In this study, we used genetics and experimental evolution of the thermophilic bacterium Thermus thermophilus to examine the ability of mutations in various components of the protein synthesis apparatus to suppress the streptomycin resistance phenotypes of mutations in ribosomal protein S12, specifically those located distant from the streptomycin binding site. With genetic selections and strain constructions, we identified suppressor mutations in EF-Tu or in ribosomal protein L11. Using experimental evolution, we identified amino acid substitutions in EF-Tu or in ribosomal proteins S4, S5, L14, or L19, some of which were found to also relieve streptomycin resistance. The wide dispersal of these mutations is consistent with long-range functional interactions among components of the translational machinery and indicates that streptomycin resistance can result from the modulation of long-range conformational signals. IMPORTANCE: The thermophilic bacterium Thermus thermophilus has become a model system for high-resolution structural studies of macromolecular complexes, such as the ribosome, while its natural competence for transformation facilitates genetic approaches. Genetic studies of T. thermophilus ribosomes can take advantage of existing high-resolution crystallographic information to allow a structural interpretation of phenotypic interactions among mutations. Using a combination of genetic selections, strain constructions, and experimental evolution, we find that certain mutations in the translation apparatus can suppress the phenotype of certain antibiotic resistance mutations. Suppression of resistance can occur by mutations located distant in the ribosome or in a translation factor. These observations suggest the existence of long-range conformational signals in the translating ribosome, particularly during the decoding of mRNA.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Estreptomicina/farmacología , Thermus thermophilus/efectos de los fármacos , Thermus thermophilus/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía , Farmacorresistencia Bacteriana , Modelos Moleculares , Mutación , Ácidos Nicotínicos , Conformación Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas , Selección Genética , Thermus thermophilus/genéticaRESUMEN
UNLABELLED: 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 α-subunit. Mutant PheS with an A294G amino acid substitution renders cells sensitive to the phenylalanine analog p-chlorophenylalanine. 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 ß-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. IMPORTANCE: Thermus 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.
Asunto(s)
ADN Bacteriano/genética , Ingeniería Genética , Genoma Bacteriano , Thermus thermophilus/genética , Alelos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Fenclonina , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica , Marcadores Genéticos , Mutación , Plásmidos/clasificación , Plásmidos/genética , ARN Bacteriano , ARN Ribosómico 16S , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The ribosome decodes mRNA by monitoring the geometry of codon-anticodon base-pairing using a set of universally conserved 16S rRNA nucleotides within the conformationally dynamic decoding site. By applying single-molecule FRET and X-ray crystallography, we have determined that conditional-lethal, streptomycin-dependence mutations in ribosomal protein S12 interfere with tRNA selection by allowing conformational distortions of the decoding site that impair GTPase activation of EF-Tu during the tRNA selection process. Distortions in the decoding site are reversed by streptomycin or by a second-site suppressor mutation in 16S rRNA. These observations encourage a refinement of the current model for decoding, wherein ribosomal protein S12 and the decoding site collaborate to optimize codon recognition and substrate discrimination during the early stages of the tRNA selection process.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Ribosómicas/química , Thermus thermophilus/genética , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli , Modelos Moleculares , Conformación de Ácido Nucleico , Mutación Puntual , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , ARN de Transferencia de Fenilalanina/química , Proteínas Ribosómicas/genética , Ribosomas/químicaRESUMEN
Thermus thermophilus is an extremely thermophilic bacterium that grows between 50 and 80 °C and is an excellent model organism not only for understanding life at high temperature but also for its biotechnological and industrial applications. Multiple molecular capabilities are available including targeted gene inactivation and the use of shuttle plasmids that replicate in T. thermophilus and Escherichia coli; however, the ability to disrupt gene function randomly by transposon insertion has not been developed. Here we report a detailed method of transposon mutagenesis of T. thermophilus HB27 based on the EZ-Tn5 system from Epicentre Biotechnologies. We were able to generate insertion mutations throughout the chromosome by in vitro transposition and transformation with mutagenized genomic DNA. We also report that an additional step, one that fills in single stranded gaps in donor DNA generated by the transposition reaction, was essential for successful mutagenesis. We anticipate that our method of transposon mutagenesis will enable further genetic development of T. thermophilus and may also be valuable for similar endeavors with other under-developed organisms.
Asunto(s)
Elementos Transponibles de ADN/genética , Mutagénesis , Thermus thermophilus/genética , Arginina/química , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Escherichia coli/genética , Fermentación , Ingeniería Genética/métodos , Plásmidos , Ácido Pirúvico/químicaRESUMEN
During protein synthesis, the ribosome undergoes conformational transitions between functional states, requiring communication between distant structural elements of the ribosome. Despite advances in ribosome structural biology, identifying the protein and rRNA residues governing these transitions remains a significant challenge. Such residues can potentially be identified genetically, given the predicted deleterious effects of mutations stabilizing the ribosome in discrete conformations and the expected ameliorating effects of second-site compensatory mutations. In this study, we employed genetic selections and experimental evolution to identify interacting mutations in the ribosome of the thermophilic bacterium Thermus thermophilus. By direct genetic selections, we identified mutations in 16S rRNA conferring a streptomycin dependence phenotype and from these derived second-site suppressor mutations relieving dependence. Using experimental evolution of streptomycin-independent pseudorevertants, we identified additional compensating mutations. Similar mutations could be evolved from slow-growing streptomycin-resistant mutants. While some mutations arose close to the site of the original mutation in the three-dimensional structure of the 30S ribosomal subunit and probably act directly by compensating for local structural distortions, the locations of others are consistent with long-range communication between specific structural elements within the ribosome.
Asunto(s)
Evolución Molecular Dirigida , Regulación Bacteriana de la Expresión Génica/fisiología , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Selección Genética , Thermus thermophilus/metabolismo , Modelos Moleculares , Mutación , Conformación Proteica , Thermus thermophilus/genéticaRESUMEN
Streptomycin is a bactericidal antibiotic that induces translational errors. It binds to the 30S ribosomal subunit, interacting with ribosomal protein S12 and with 16S rRNA through contacts with the phosphodiester backbone. To explore the structural basis for streptomycin resistance, we determined the X-ray crystal structures of 30S ribosomal subunits from six streptomycin-resistant mutants of Thermus thermophilus both in the apo form and in complex with streptomycin. Base substitutions at highly conserved residues in the central pseudoknot of 16S rRNA produce novel hydrogen-bonding and base-stacking interactions. These rearrangements in secondary structure produce only minor adjustments in the three-dimensional fold of the pseudoknot. These results illustrate how antibiotic resistance can occur as a result of small changes in binding site conformation.
Asunto(s)
Farmacorresistencia Bacteriana/genética , Mutación Puntual , ARN Ribosómico 16S/metabolismo , Proteínas Ribosómicas/química , Subunidades Ribosómicas Pequeñas Bacterianas/química , Antibacterianos/química , Antibacterianos/farmacología , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Biosíntesis de Proteínas/efectos de los fármacos , ARN Ribosómico 16S/química , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/efectos de los fármacos , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Estreptomicina/química , Estreptomicina/farmacología , Thermus thermophilus/química , Thermus thermophilus/efectos de los fármacos , Thermus thermophilus/genéticaRESUMEN
High-resolution ribosome structures determined by X-ray crystallography have provided important insights into the mechanism of translation. Such studies have thus far relied on large ribosome crystals kept at cryogenic temperatures to reduce radiation damage. Here, the application of serial femtosecond X-ray crystallography (SFX) using an X-ray free-electron laser (XFEL) to obtain diffraction data from ribosome microcrystals in liquid suspension at ambient temperature is described. 30S ribosomal subunit microcrystals diffracted to beyond 6â Å resolution, demonstrating the feasibility of using SFX for ribosome structural studies. The ability to collect diffraction data at near-physiological temperatures promises to provide fundamental insights into the structural dynamics of the ribosome and its functional complexes.
Asunto(s)
Electrones , Subunidades Ribosómicas Pequeñas Bacterianas/ultraestructura , Thermus thermophilus/química , Cristalización , Cristalografía por Rayos X , Rayos Láser , Subunidades Ribosómicas Pequeñas Bacterianas/química , Temperatura , Difracción de Rayos XRESUMEN
The ribosome is an essential drug target as many classes of clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent mechanisms of resistance to PTC-acting drugs is C8-methylation of the universally conserved adenine residue 2503 (A2503) of the 23S rRNA by the methyltransferase Cfr. Despite its clinical significance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. In this work, we developed a method to express a functionally-active Cfr-methyltransferase in the thermophilic bacterium Thermus thermophilus and report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-tRNAs. Our structures reveal that an allosteric rearrangement of nucleotide A2062 upon Cfr-methylation of A2503 is likely responsible for the inability of some PTC inhibitors to bind to the ribosome, providing additional insights into the Cfr resistance mechanism. Lastly, by determining the structures of the Cfr-methylated ribosome in complex with the antibiotics iboxamycin and tylosin, we provide the structural bases behind two distinct mechanisms of evading Cfr-mediated resistance.
RESUMEN
All organisms incorporate post-transcriptional modifications into ribosomal RNA, influencing ribosome assembly and function in ways that are poorly understood. The most highly conserved modification is the dimethylation of two adenosines near the 3' end of the small subunit rRNA. Lack of these methylations due to deficiency in the KsgA methyltransferase stimulates translational errors during both the initiation and elongation phases of protein synthesis and confers resistance to the antibiotic kasugamycin. Here, we present the X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit lacking these dimethylations. Our data indicate that the KsgA-directed methylations facilitate structural rearrangements in order to establish a functionally optimum subunit conformation during the final stages of ribosome assembly.
Asunto(s)
Metiltransferasas/metabolismo , ARN Ribosómico 16S/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/química , Ribosomas/fisiología , Secuencia de Bases , Cristalografía por Rayos X , Metilación , Metiltransferasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformación de Ácido Nucleico , Conformación Proteica , ARN Ribosómico 16S/química , ARN Ribosómico 16S/fisiología , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/fisiología , Ribosomas/química , Ribosomas/metabolismo , Relación Estructura-Actividad , Thermus thermophilus/química , Thermus thermophilus/metabolismo , Thermus thermophilus/fisiologíaRESUMEN
Cells devote a significant effort toward the production of multiple modified nucleotides in rRNAs, which fine tune the ribosome function. Here, we report that two methyltransferases, RsmB and RsmF, are responsible for all four 5-methylcytidine (m(5)C) modifications in 16S rRNA of Thermus thermophilus. Like Escherichia coli RsmB, T. thermophilus RsmB produces m(5)C967. In contrast to E. coli RsmF, which introduces a single m(5)C1407 modification, T. thermophilus RsmF modifies three positions, generating m(5)C1400 and m(5)C1404 in addition to m(5)C1407. These three residues are clustered near the decoding site of the ribosome, but are situated in distinct structural contexts, suggesting a requirement for flexibility in the RsmF active site that is absent from the E. coli enzyme. Two of these residues, C1400 and C1404, are sufficiently buried in the mature ribosome structure so as to require extensive unfolding of the rRNA to be accessible to RsmF. In vitro, T. thermophilus RsmF methylates C1400, C1404, and C1407 in a 30S subunit substrate, but only C1400 and C1404 when naked 16S rRNA is the substrate. The multispecificity of T. thermophilus RsmF is potentially explained by three crystal structures of the enzyme in a complex with cofactor S-adenosyl-methionine at up to 1.3 A resolution. In addition to confirming the overall structural similarity to E. coli RsmF, these structures also reveal that key segments in the active site are likely to be dynamic in solution, thereby expanding substrate recognition by T. thermophilus RsmF.
Asunto(s)
Metiltransferasas/química , Metiltransferasas/metabolismo , ARN Ribosómico/metabolismo , Thermus thermophilus/enzimología , Thermus thermophilus/genética , Sitios de Unión/genética , Citidina/análogos & derivados , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Metiltransferasas/genética , Nucleótidos/química , Nucleótidos/genética , ARN Ribosómico/genética , Ribosomas/genética , Ribosomas/metabolismo , Thermus thermophilus/metabolismoRESUMEN
The Escherichia coli rluD gene encodes a pseudouridine synthase responsible for the pseudouridine (Ψ) 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 Ψ 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 Ψ modifications.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Hidroliasas/metabolismo , Ribosomas/metabolismo , Salmonella enterica/metabolismo , Alelos , Secuencia de Bases , Escherichia coli/clasificación , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Modelos Moleculares , Mutación , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Fenotipo , Conformación Proteica , Seudouridina/análogos & derivados , Seudouridina/genética , Seudouridina/metabolismo , Ribosomas/genética , Salmonella enterica/clasificación , Salmonella enterica/genéticaRESUMEN
Characterization of base substitutions in rRNAs has provided important insights into the mechanism of protein synthesis. Knowledge of the structural effects of such alterations is limited, and could be greatly expanded with the development of a genetic system based on an organism amenable to both genetics and structural biology. Here, we describe the genetic analysis of base substitutions in 16S ribosomal RNA of the extreme thermophile Thermus thermophilus, and an analysis of the conformational effects of these substitutions by structure probing with base-specific modifying agents. Gene replacement methods were used to construct a derivative of strain HB8 carrying a single 16S rRNA gene, allowing the isolation of spontaneous streptomycin-resistant mutants and subsequent genetic mapping of mutations by recombination. The residues altered to give streptomycin resistance reside within the central pseudoknot structure of 16S rRNA comprised of helices 1 and 27, and participate in the U13-U20-A915 base triple, the G21-A914 type II sheared G-A base pair, or the G885-C912 Watson-Crick base pair closing helix 27. Substitutions at any of the three residues engaged in the base triple were found to confer resistance. Results from structure probing of the pseudoknot are consistent with perturbation of RNA conformation by these substitutions, potentially explaining their streptomycin-resistance phenotypes.
Asunto(s)
Farmacorresistencia Bacteriana/genética , Conformación de Ácido Nucleico , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Thermus thermophilus/genética , Sustitución de Aminoácidos , Secuencia de Bases , Técnicas de Inactivación de Genes , Mutación , Biosíntesis de Proteínas/genética , ARN Bacteriano/química , ARN Ribosómico 16S/química , Proteínas Ribosómicas/genética , Análisis de Secuencia de ARN , Estreptomicina/farmacología , Thermus thermophilus/efectos de los fármacosRESUMEN
Codon recognition by aminoacyl-tRNA on the ribosome triggers a process leading to GTP hydrolysis by elongation factor Tu (EF-Tu) and release of aminoacyl-tRNA into the A site of the ribosome. The nature of this signal is largely unknown. Here, we present genetic evidence that a specific set of direct interactions between ribosomal protein S12 and aminoacyl-tRNA, together with contacts between S12 and 16S rRNA, provide a pathway for the signaling of codon recognition to EF-Tu. Three novel amino acid substitutions, H76R, R37C, and K53E in Thermus thermophilus ribosomal protein S12, confer resistance to streptomycin. The streptomycin-resistance phenotypes of H76R, R37C, and K53E are all abolished by the mutation A375T in EF-Tu. A375T confers resistance to kirromycin, an antibiotic freezing EF-Tu in a GTPase activated state. H76 contacts aminoacyl-tRNA in ternary complex with EF-Tu and GTP, while R37 and K53 are involved in the conformational transition of the 30S subunit occurring upon codon recognition. We propose that codon recognition and domain closure of the 30S subunit are signaled through aminoacyl-tRNA to EF-Tu via these S12 residues.
Asunto(s)
Proteínas Bacterianas/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Proteínas Ribosómicas/metabolismo , Thermus thermophilus/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Codón/genética , Codón/metabolismo , Farmacorresistencia Bacteriana/genética , Factor Tu de Elongación Peptídica/genética , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Conformación Proteica , Piridonas/farmacología , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Estreptomicina/farmacología , Thermus thermophilus/genéticaRESUMEN
The RsmG methyltransferase is responsible for N(7) methylation of G527 of 16S rRNA in bacteria. Here, we report the identification of the Thermus thermophilus rsmG gene, the isolation of rsmG mutants, and the solution of RsmG X-ray crystal structures at up to 1.5 A resolution. Like their counterparts in other species, T. thermophilus rsmG mutants are weakly resistant to the aminoglycoside antibiotic streptomycin. Growth competition experiments indicate a physiological cost to loss of RsmG activity, consistent with the conservation of the modification site in the decoding region of the ribosome. In contrast to Escherichia coli RsmG, which has been reported to recognize only intact 30S subunits, T. thermophilus RsmG shows no in vitro methylation activity against native 30S subunits, only low activity with 30S subunits at low magnesium concentration, and maximum activity with deproteinized 16S rRNA. Cofactor-bound crystal structures of RsmG reveal a positively charged surface area remote from the active site that binds an adenosine monophosphate molecule. We conclude that an early assembly intermediate is the most likely candidate for the biological substrate of RsmG.
Asunto(s)
ARN Ribosómico 16S/metabolismo , Thermus thermophilus/enzimología , ARNt Metiltransferasas/química , ARNt Metiltransferasas/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Dominio Catalítico , Cristalografía por Rayos X , Farmacorresistencia Bacteriana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , Conformación de Ácido Nucleico , Organismos Modificados Genéticamente , Fenotipo , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Homología de Secuencia de Aminoácido , Estreptomicina/metabolismo , Thermus thermophilus/genética , Thermus thermophilus/aislamiento & purificación , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismoRESUMEN
Rhodothermus marinus is a halophilic extreme thermophile, with potential as a model organism for studies of the structural basis of antibiotic resistance. In order to facilitate genetic studies of this organism, we have surveyed the antibiotic sensitivity spectrum of R. marinus and identified spontaneous antibiotic-resistant mutants. R. marinus is naturally insensitive to aminoglycosides, aminocylitols and tuberactinomycins that target the 30S ribosomal subunit, but is sensitive to all 50S ribosomal subunit-targeting antibiotics examined, including macrolides, lincosamides, streptogramin B, chloramphenicol, and thiostrepton. It is also sensitive to kirromycin and fusidic acid, which target protein synthesis factors. It is sensitive to rifampicin (RNA polymerase inhibitor) and to the fluoroquinolones ofloxacin and ciprofloxacin (DNA gyrase inhibitors), but insensitive to nalidixic acid. Drug-resistant mutants were identified using rifampicin, thiostrepton, erythromycin, spiramycin, tylosin, lincomycin, and chloramphenicol. The majority of these were found to have mutations that are similar or identical to those previously found in other species, while several novel mutations were identified. This study provides potential selectable markers for genetic manipulations and demonstrates the feasibility of using R. marinus as a model system for studies of ribosome and RNA polymerase structure, function, and evolution.