RESUMEN
30S subunits become inactive upon exposure to low Mg2+ concentration, because of a reversible conformational change that entails nucleotides (nt) in the neck helix (h28) and 3' tail of 16S rRNA. This active-to-inactive transition involves partial unwinding of h28 and repairing of nt 921-923 with nt 1532-1534, which requires flipping of the 3' tail by â¼180°. Growing evidence suggests that immature 30S particles adopt the inactive conformation in the cell, and transition to the active state occurs at a late stage of maturation. Here, we target nucleotides that form the alternative helix (hALT) of the inactive state. Using an orthogonal ribosome system, we find that disruption of hALT decreases translation activity in the cell modestly, by approximately twofold, without compromising ribosome fidelity. Ribosomes carrying substitutions at positions 1532-1533 support the growth of Escherichia coli strain Δ7 prrn (which carries a single rRNA operon), albeit at rates 10%-20% slower than wild-type ribosomes. These mutant Δ7 prrn strains accumulate free 30S particles and precursor 17S rRNA, indicative of biogenesis defects. Analysis of purified control and mutant subunits suggests that hALT stabilizes the inactive state by 1.2 kcal/mol with little-to-no impact on the active state or the transition state of conversion.
Asunto(s)
Escherichia coli , Conformación de Ácido Nucleico , ARN Ribosómico 16S , Subunidades Ribosómicas Pequeñas Bacterianas , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Biosíntesis de Proteínas , Magnesio/metabolismoRESUMEN
Neisseria commensals are an indisputable source of resistance for their pathogenic relatives. However, the evolutionary paths commensal species take to reduced susceptibility in this genus have been relatively underexplored. Here, we leverage in vitro selection as a powerful screen to identify the genetic adaptations that produce azithromycin resistance (≥ 2 µg/mL) in the Neisseria commensal, N. elongata. Across multiple lineages (n = 7/16), we find mutations that reduce susceptibility to azithromycin converge on the locus encoding the 50S ribosomal L34 protein (rpmH) and the intergenic region proximal to the 30S ribosomal S3 protein (rpsC) through short tandem duplication events. Interestingly, one of the laboratory evolved mutations in rpmH is identical (7LKRTYQ12), and two nearly identical, to those recently reported to contribute to high-level azithromycin resistance in N. gonorrhoeae. Transformations into the ancestral N. elongata lineage confirmed the causality of both rpmH and rpsC mutations. Though most lineages inheriting duplications suffered in vitro fitness costs, one variant showed no growth defect, suggesting the possibility that it may be sustained in natural populations. Ultimately, studies like this will be critical for predicting commensal alleles that could rapidly disseminate into pathogen populations via allelic exchange across recombinogenic microbial genera.
Asunto(s)
Farmacorresistencia Bacteriana/genética , Macrólidos/farmacología , Neisseria/genética , Antibacterianos/farmacología , Azitromicina/farmacología , Pruebas de Sensibilidad Microbiana , Microbiota/genética , Inhibidores de la Síntesis de la Proteína , ARN Ribosómico 23S/genética , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Grandes Bacterianas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Ribosomas/genética , Eliminación de Secuencia/genéticaRESUMEN
Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.
Asunto(s)
Biosíntesis de Proteínas , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/química , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Ribosomas/química , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismoRESUMEN
The multi-domain bacterial S1 protein is the largest and most functionally important ribosomal protein of the 30S subunit, which interacts with both mRNA and proteins. The family of ribosomal S1 proteins differs in the classical sense from a protein with tandem repeats and has a "bead-on-string" organization, where each repeat is folded into a globular domain. Based on our recent data, the study of evolutionary relationships for the bacterial phyla will provide evidence for one of the proposed theories of the evolutionary development of proteins with structural repeats: from multiple repeats of assembles to single repeats, or vice versa. In this comparative analysis of 1333 S1 sequences that were identified in 24 different phyla, we demonstrate how such phyla can form independently/dependently during evolution. To the best of our knowledge, this work is the first study of the evolutionary history of bacterial ribosomal S1 proteins. The collected and structured data can be useful to computer biologists as a resource for determining percent identity, amino acid composition and logo motifs, as well as dN/dS ratio in bacterial S1 protein. The obtained research data indicate that the evolutionary development of bacterial ribosomal S1 proteins evolved from multiple assemblies to single repeat. The presented data are integrated into the server, which can be accessed at http://oka.protres.ru:4200.
Asunto(s)
Algoritmos , Bacterias/genética , Evolución Biológica , Genoma Bacteriano , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Bacterias/clasificación , Bacterias/metabolismo , Conjuntos de Datos como Asunto , Expresión Génica , Metagenoma , Filogenia , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Programas InformáticosRESUMEN
The ribosomal protein uS12 is conserved across all domains of life. Recently, a heterozygous spontaneous mutation in human uS12 (corresponding to R49K mutation immediately downstream of the universally conserved 44 PNSA47 loop in Escherichia coli uS12) was identified for causing ribosomopathy, highlighting the importance of the PNSA loop. To investigate the effects of a similar mutation in the absence of any wild-type alleles, we mutated the rpsL gene (encoding uS12) in E. coli. Consistent with its pathology (in humans), we were unable to generate the R49K mutation in E. coli in the absence of a support plasmid. However, we were able to generate the L48K mutation in its immediate vicinity. The L48K mutation resulted in a cold sensitive phenotype and ribosome biogenesis defect in the strain. We show that the L48K mutation impacts the steps of initiation and elongation. Furthermore, the genetic interactions of the L48K mutation with RRF and Pth suggest a novel role of the PNSA loop in ribosome recycling. Our studies reveal new functions of the PNSA loop in uS12, which has so far been studied in the context of translation elongation.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Extensión de la Cadena Peptídica de Translación/genética , Iniciación de la Cadena Peptídica Traduccional/genética , Proteínas Ribosómicas/genética , Escherichia coli/metabolismo , Humanos , Conformación Proteica , ARN Ribosómico 16S/genética , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismoRESUMEN
In response to DNA damage, Escherichia coli cells activate the expression of the toxin gene tisB of the toxin-antitoxin system tisB-istR1. Of three isoforms, only the processed, highly structured +42 tisB mRNA is active. Translation requires a standby site, composed of two essential elements: a single-stranded region located 100 nucleotides upstream of the sequestered RBS, and a structure near the 5'-end of the active mRNA. Here, we propose that this 5'-structure is an RNA pseudoknot which is required for 30S and protein S1-alone binding to the mRNA. Point mutations that prevent formation of this pseudoknot inhibit formation of translation initiation complexes, impair S1 and 30S binding to the mRNA, and render the tisB mRNA non-toxic in vivo. A set of mutations created in either the left or right arm of stem 2 of the pseudoknot entailed loss of toxicity upon overexpression of the corresponding mRNA variants. Combining the matching right-left arm mutations entirely restored toxicity levels to that of the wild-type, active mRNA. Finally, since many pseudoknots have high affinity for S1, we predicted similar pseudoknots in non-homologous type I toxin-antitoxin systems that exhibit features similar to that of tisB-IstR1, suggesting a shared requirement for standby acting at great distances.
Asunto(s)
Toxinas Bacterianas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Biosíntesis de Proteínas , ARN Bacteriano/genética , ARN Mensajero/genética , Sistemas Toxina-Antitoxina/genética , Toxinas Bacterianas/metabolismo , Emparejamiento Base , Secuencia de Bases , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Conformación de Ácido Nucleico , Mutación Puntual , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismoRESUMEN
BACKGROUND: Our previous research indicated the excellent in vitro antibacterial activity of Eravacycline (Erava) and its heteroresistance frequency against clinical Staphylococcus aureus isolates. In this study, we further aimed to investigate the mechanisms of Erava resistance and heteroresistance in S. aureus. Eight parental S. aureus isolates were induced under Erava pressure in vitro and the Erava-resistant isolates were selected and identified. Then, the genetic mutations of 30S ribosomal subunits were analyzed by PCR and sequence alignment. RT-qPCR analysis were performed to compare the relative expression of eight candidate genes impacting the susceptibility of tetracycline (Tet) between the resistant or heteroresistant and parental isolates. Furthermore, the in vitro overexpression vectors of three selected candidate genes were constructed to test their impact on the heteroresistance and resistance of Erava in S. aureus. RESULTS: The MICs elevation in Erava-induced resistant S. aureus isolates were identified and the increasing MICs values of another two Tet class antibiotics, including both omadacycline (Omada) and tigecycline (Tige) were also tested. Genetic mutations in 30S ribosomal protein S10 were found frequently in Erava-derived resistant isolates. RT-qPCR analysis and the in vitro overexpression experiments indicated that USA300HOU_RS00550 (an Na/Pi cotransporter family protein) and USA300HOU_RS01625 (a branched-chain amino acid transport system II carrier protein) contributed to Erava heteroresistance in S. aureus. CONCLUSION: Genetic mutation of 30S ribosome subunits contributed to Erava resistance, and the transcriptional overexpression of USA300HOU_RS01625 and USA300HOU_RS00550 also participated in the occurrence of Erava heteroresistance in S. aureus.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Tetraciclinas/farmacología , China , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificación , Tigeciclina/farmacologíaRESUMEN
In vitro reconstitution is a powerful tool for investigating ribosome functions and biogenesis, as well as discovering new ribosomal features. In this study, we integrated all of the processes required for Escherichia coli small ribosomal subunit assembly. In our method, termed fully Recombinant-based integrated Synthesis, Assembly, and Translation (R-iSAT), assembly and evaluation of the small ribosomal subunits are coupled with ribosomal RNA (rRNA) synthesis in a reconstituted cell-free protein synthesis system. By changing the components of R-iSAT, including recombinant ribosomal protein composition, we coupled ribosomal assembly with ribosomal protein synthesis, enabling functional synthesis of ribosomal proteins and subsequent subunit assembly. In addition, we assembled and evaluated subunits with mutations in both rRNA and ribosomal proteins. The study demonstrated that our scheme provides new ways to comprehensively analyze any elements of the small ribosomal subunit, with the goal of improving our understanding of ribosomal biogenesis, function, and engineering.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , ARN Bacteriano/metabolismo , ARN Ribosómico 16S/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Transcripción Genética , Proteínas Bacterianas/genética , Escherichia coli/genética , Mutación , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/genéticaRESUMEN
This study aimed to evaluate the in vitro antimicrobial activity, heteroresistance emergence, and resistance mechanism of omadacycline (OMC) in clinical Enterococcus faecalis isolates from China. A total of 276 isolates were collected retrospectively in China from 2011 to 2015. The MICs of OMC, doxycycline (DOX), and minocycline (MIN) against E. faecalis were determined by broth microdilution. Tetracycline (TET)-specific resistance genes and multilocus sequence typing (MLST) of the isolates were investigated using PCR. The detection frequency of OMC heteroresistance in E. faecalis was evaluated with population analysis profiling (PAP). The mechanism of OMC heteroresistance and resistance in E. faecalis was examined by amplifying 30S ribosomal subunit genes, RNA sequencing (RNA-Seq), and in vitro recombination experiments. The OMC MICs of clinical E. faecalis isolates ranged from ≤0.06 to 1.0 mg/liter, and 42% of the E. faecalis isolates with an OMC MIC of 1.0 mg/liter were found to be sequence type 16 (ST16). Six OMC-heteroresistant isolates with MIC values of ≤0.5 mg/liter were detected among 238 E. faecalis isolates. The resistant subpopulations of heteroresistant isolates showed OMC MICs in the range of 2 to 4 mg/liter and were found without 30S ribosomal subunit gene mutations. Moreover, RNA sequencing and in vitro recombination experiments demonstrated that overexpression of a bone morphogenetic protein (BMP) family ATP-binding cassette (ABC) transporter substrate-binding protein, OG1RF_RS00630, facilitated OMC heteroresistance in E. faecalis In conclusion, OMC exhibited better activity against clinical E. faecalis isolates from China than that of DOX or MIN, and overexpression of OG1RF_RS00630 in E. faecalis facilitated the development of OMC heteroresistance.
Asunto(s)
Antibacterianos/farmacología , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/genética , Tetraciclinas/farmacología , China , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Mutación/genética , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Análisis de Secuencia de ARNRESUMEN
Dityromycin is a peptide antibiotic isolated from the culture broth of the soil microorganism Streptomyces sp. strain AM-2504. Recent structural studies have shown that dityromycin targets the ribosomal protein S12 in the 30S ribosomal subunit, inhibiting translocation. Herein, by using in vitro protein synthesis assays, we identified the resistance mechanism of the producer strain to the secondary metabolite dityromycin. The results show that the self-resistance mechanism of the Streptomyces sp. strain AM-2504 is due to a specific modification of the ribosome. In particular, two amino acid substitutions, located in a highly conserved region of the S12 protein corresponding to the binding site of the antibiotic, were found. These mutations cause a substantial loss of affinity of the dityromycin for the 30S ribosomal subunit, protecting the producer strain from the toxic effect of the antibiotic. In addition to providing a detailed description of the first mechanism of self-resistance based on a mutated ribosomal protein, this work demonstrates that the molecular determinants of the dityromycin resistance identified in Streptomyces can be transferred to Escherichia coli ribosomes, where they can trigger the same antibiotic resistance mechanism found in the producer strain.IMPORTANCE The World Health Organization has identified antimicrobial resistance as a substantial threat to human health. Because of the emergence of pathogenic bacteria resistant to multiple antibiotics worldwide, there is a need to identify the mode of action of antibiotics and to unravel the basic mechanisms responsible for drug resistance. Antibiotic producers' microorganisms can protect themselves from the toxic effect of the drug using different strategies; one of the most common involves the modification of the antibiotic's target site. In this work, we report a detailed analysis of the molecular mechanism, based on protein modification, devised by the soil microorganism Streptomyces sp. strain AM-2504 to protect itself from the activity of the peptide antibiotic dityromycin. Furthermore, we demonstrate that this mechanism can be reproduced in E. coli, thereby eliciting antibiotic resistance in this human commensal bacterium.
Asunto(s)
Antibacterianos/farmacología , Depsipéptidos/farmacología , Farmacorresistencia Bacteriana , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Streptomyces/efectos de los fármacos , Sustitución de Aminoácidos , Sitios de Unión , Depsipéptidos/biosíntesis , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Mutagénesis Sitio-Dirigida , Biosíntesis de Proteínas , Dominios y Motivos de Interacción de Proteínas , Proteínas Ribosómicas/genética , Metabolismo Secundario , Streptomyces/genéticaRESUMEN
Ribosome assembly cofactors are widely conserved across all domains of life. One such group, the ribosome-associated GTPases (RA-GTPase), act as molecular switches to coordinate ribosome assembly. We previously identified the Staphylococcus aureus RA-GTPase Era as a target for the stringent response alarmone (p)ppGpp, with binding leading to inhibition of GTPase activity. Era is highly conserved throughout the bacterial kingdom and is essential in many species, although the function of Era in ribosome assembly is unclear. Here we show that Era is not essential in S. aureus but is important for 30S ribosomal subunit assembly. Protein interaction studies reveal that Era interacts with the 16S rRNA endonuclease YbeY and the DEAD-box RNA helicase CshA. We determine that both Era and CshA are required for growth at suboptimal temperatures and rRNA processing. Era and CshA also form direct interactions with the (p)ppGpp synthetase RelSau, with RelSau positively impacting the GTPase activity of Era but negatively affecting the helicase activity of CshA. We propose that in its GTP-bound form, Era acts as a hub protein on the ribosome to direct enzymes involved in rRNA processing/degradation and ribosome subunit assembly to their site of action. This activity is impeded by multiple components of the stringent response, contributing to the slowed growth phenotype synonymous with this stress response pathway.
Asunto(s)
Aclimatación/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al GTP/metabolismo , ARN Ribosómico/metabolismo , Staphylococcus aureus/fisiología , Frío/efectos adversos , ARN Helicasas DEAD-box/metabolismo , Endonucleasas/metabolismo , Ligasas/metabolismo , Biogénesis de Organelos , Unión Proteica/fisiología , Mapeo de Interacción de Proteínas , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismoRESUMEN
Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is important to expand on studies of 30S assembly intermediates purified from bacterial strains lacking particular maturation factors. To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, high resolution cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Era's role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Proteínas de Unión al GTP/metabolismo , Homeostasis , ARN Ribosómico 16S/metabolismo , Proteínas de Unión al ARN/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Subunidades Ribosómicas Pequeñas/metabolismo , Secuencia de Bases , Sitios de Unión , Microscopía por Crioelectrón , Proteínas de Escherichia coli/genética , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/genética , Conformación de Ácido Nucleico , Unión Proteica , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genética , Proteínas de Unión al ARN/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas/genética , Subunidades Ribosómicas Pequeñas/ultraestructura , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/ultraestructuraRESUMEN
rRNAs and tRNAs universally require processing from longer primary transcripts to become functional for translation. Here, we describe an unsuspected link between tRNA maturation and the 3' processing of 16S rRNA, a key step in preparing the small ribosomal subunit for interaction with the Shine-Dalgarno sequence in prokaryotic translation initiation. We show that an accumulation of either 5' or 3' immature tRNAs triggers RelA-dependent production of the stringent response alarmone (p)ppGpp in the Gram-positive model organism Bacillus subtilis. The accumulation of (p)ppGpp and accompanying decrease in GTP levels specifically inhibit 16S rRNA 3' maturation. We suggest that cells can exploit this mechanism to sense potential slowdowns in tRNA maturation and adjust rRNA processing accordingly to maintain the appropriate functional balance between these two major components of the translation apparatus.
Asunto(s)
Bacillus subtilis/genética , Regulación Bacteriana de la Expresión Génica , Guanosina Pentafosfato/biosíntesis , Iniciación de la Cadena Peptídica Traduccional , ARN Ribosómico 16S/genética , ARN de Transferencia/genética , Bacillus subtilis/metabolismo , Secuencia de Bases , Guanosina Pentafosfato/genética , Guanosina Trifosfato/metabolismo , Ligasas/genética , Ligasas/metabolismo , Conformación de Ácido Nucleico , ARN Ribosómico 16S/química , ARN Ribosómico 16S/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Subunidades Ribosómicas Grandes Bacterianas/genética , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismoRESUMEN
Tiancimycins (TNMs) are a group of 10-membered anthraquinone-fused enediynes, newly discovered from Streptomyces sp. CB03234. Among them, TNM-A and TNM-D have exhibited excellent antitumor performances and could be exploited as very promising warheads for the development of anticancer antibody-drug conjugates (ADCs). However, their low titers, especially TNM-D, have severely limited following progress. Therefore, the streptomycin-induced ribosome engineering was adopted in this work for strain improvement of CB03234, and a TNMs high producer S. sp. CB03234-S with the K43N mutation at 30S ribosomal protein S12 was successfully screened out. Subsequent media optimization revealed the essential effects of iodide and copper ion on the production of TNMs, while the substitution of nitrogen source could evidently promote the accumulation of TNM-D, and the ratio of produced TNM-A and TNM-D was responsive to the change of carbon and nitrogen ratio in the medium. Further amelioration of the pH control in scaled up 25 L fermentation increased the average titers of TNM-A and TNM-D up to 13.7 ± 0.3 and 19.2 ± 0.4 mg/L, respectively. The achieved over 45-fold titer improvement of TNM-A, and 109-fold total titer improvement of TNM-A and TNM-D enabled the efficient purification of over 200 mg of each target molecule from 25 L fermentation. Our efforts have demonstrated a practical strategy for titer improvement of anthraquinone-fused enediynes and set up a solid base for the pilot scale production and preclinical studies of TNMs to expedite the future development of anticancer ADC drugs.
Asunto(s)
Enediinos , Fermentación/genética , Ingeniería Metabólica/métodos , Ribosomas , Estreptomicina/farmacología , Antineoplásicos/análisis , Antineoplásicos/química , Antineoplásicos/metabolismo , Enediinos/análisis , Enediinos/química , Enediinos/metabolismo , Mutación/genética , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Streptomyces/efectos de los fármacos , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
S1, a multi-domain ribosomal protein associated with the 30S subunit, is essential for translation initiation. S1 binds with high affinity to single-stranded mRNA containing A/U-rich patches upstream of the start codon. It was previously reported that domains 1-3 of S1 protein play a role in the docking and unfolding of structured mRNAs to the ribosome. Moreover, S1-deficient 30S subunits are still able to bind to low structured mRNAs. However, mRNAs containing A/U-rich patches in the early base positions after start codon enhance protein synthesis and mRNA binding to the ribosome, which suggests that S1 is also able to interact with these A/U-rich regions. To evaluate the essentiality of S1 domains in the binding to low structured mRNAs containing A/U/G nucleotides after the start codon as well as their role in translation and cell viability, S1 protein deletion variants were generated. We show that S1 domain 3 is necessary to discriminate these mRNAs according to the nucleotide nature since its absence abrogated S1 binding to A/U-rich mRNAs and allowed binding to G-rich mRNAs. Interestingly, domains 2 and 3 were required for the binding of mRNAs containing A/U-rich sequences after the start codon to 30S, in vitro translation and cell viability.
Asunto(s)
Escherichia coli/química , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/química , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Animales , Femenino , Ratas , Ratas Wistar , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/genéticaRESUMEN
Initiator tRNAs (i-tRNAs) possess highly conserved three consecutive GC base pairs (GC/GC/GC, 3GC pairs) in their anticodon stems. Additionally, in bacteria and eukaryotic organelles, the amino acid attached to i-tRNA is formylated by Fmt to facilitate its targeting to 30S ribosomes. Mutations in GC/GC/GC to UA/CG/AU in i-tRNACUA/3GC do not affect its formylation. However, the i-tRNACUA/3GC is non-functional in initiation. Here, we characterised an Escherichia coli strain possessing an amber mutation in its fmt gene (fmtam274), which affords initiation with i-tRNACUA/3GC. Replacement of fmt with fmtam274 in the parent strain results in production of truncated Fmt, accumulation of unformylated i-tRNA, and a slow growth phenotype. Introduction of i-tRNACUA/3GC into the fmtam274 strain restores accumulation of formylated i-tRNAs and rescues the growth defect of the strain. We show that i-tRNACUA/3GC causes a low level suppression of am274 in fmtam274. Low levels of cellular Fmt lead to compromised efficiency of formylation of i-tRNAs, which in turn results in distribution of the charged i-tRNAs between IF2 and EF-Tu allowing the plasmid borne i-tRNACUA/3GC to function at both the initiation and elongation steps. We show that a speedy formylation of i-tRNA population is crucial for its preferential binding (and preventing other tRNAs) into the P-site.
Asunto(s)
Anticodón/genética , Conformación de Ácido Nucleico , ARN de Transferencia de Metionina/química , Ribosomas/química , Anticodón/química , Escherichia coli/química , Escherichia coli/genética , Plásmidos/genética , ARN de Transferencia de Metionina/genética , Subunidades Ribosómicas Pequeñas Bacterianas/química , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Ribosomas/genéticaRESUMEN
Among different RNA modifications, the helix 69 (H69) region of the bacterial ribosomal RNA (rRNA) contains three pseudouridines (Ψs). H69 is functionally important due to its location in the heart of the ribosome. Several structural and functional studies have shown the importance of Ψ modifications in influencing the H69 conformation as well as maintaining key interactions in the ribosome during protein synthesis. Therefore, a need exists to understand the influence of modified nucleosides on conformational dynamics of the ribosome under solution conditions that mimic the cellular environment. In this review on chemical probing, we provide detailed protocols for the use of dimethyl sulfate (DMS) to examine H69 conformational states and the influence of Ψ modifications under varying solution conditions in the context of both ribosomal subunits and full ribosomes. The use of DMS footprinting to study the binding of aminoglycosides to the H69 region of bacterial rRNA as a potential antibiotic target will also be discussed. As highlighted in this work, DMS probing and footprinting are versatile techniques that can be used to gain important insight into RNA local structure and RNA-ligand interactions, respectively.
Asunto(s)
Escherichia coli/genética , Impresión Molecular/métodos , Seudouridina/química , ARN Ribosómico 16S/química , ARN Ribosómico 23S/química , Compuestos de Anilina/química , Antibacterianos/farmacología , Fraccionamiento Celular/métodos , ADN Complementario/biosíntesis , ADN Complementario/química , ADN Complementario/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Gentamicinas/farmacología , Hidroliasas/genética , Hidroliasas/metabolismo , Ligandos , Cloruro de Magnesio/farmacología , Neomicina/farmacología , Conformación de Ácido Nucleico , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Seudouridina/genética , Seudouridina/metabolismo , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/metabolismo , Transcripción Reversa , Subunidades Ribosómicas Grandes Bacterianas/química , Subunidades Ribosómicas Grandes Bacterianas/efectos de los fármacos , Subunidades Ribosómicas Grandes Bacterianas/genética , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/química , Subunidades Ribosómicas Pequeñas Bacterianas/efectos de los fármacos , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Ribosomas/química , Ribosomas/efectos de los fármacos , Ribosomas/genética , Ribosomas/metabolismo , Ésteres del Ácido Sulfúrico/químicaRESUMEN
The Sra protein is a component of the 30S ribosomal subunit while RimJ is a ribosome-associated protein that plays a role in the maturation of the 30S ribosomal subunit. Here we found that Δsra and ΔrimJ cells showed a delayed initiation of DNA replication, prolonged doubling time, decreased cell size, and decreased amounts of total protein and DnaA per cell compared with these observed for wild-type cells. A temperature sensitivity test demonstrated that absence of the Sra or RimJ protein did not change the temperature sensitivity of the dnaA46, dnaB252, or dnaC2 mutants. Moreover, ectopic expression of Sra reversed the mutant phenotype while cells carrying the pACYC177-rimJ plasmid did not reverse the rimJ mutant phenotype. The results indicate that deletion of sra or rimJ cause defects in ribosomal function and affect the translation process, leading to a decrease in synthesis of proteins including DnaA. Therefore, we conclude that Sra- and RimJ-mediated ribosomal function is required for precise timing of initiation of chromosome replication.
Asunto(s)
Cromosomas Bacterianos/metabolismo , Replicación del ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Proteínas Bacterianas/biosíntesis , Tamaño de la Célula , Proteínas de Unión al ADN/biosíntesis , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Eliminación de Gen , Fenotipo , Biosíntesis de Proteínas , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/genéticaRESUMEN
The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New light sources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit decoding complex to 3.45 Å resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation to structural studies of RNA and RNA-protein complexes at near-physiological temperatures.
Asunto(s)
Sustancias Macromoleculares/química , Conformación de Ácido Nucleico , Subunidades Ribosómicas Pequeñas Bacterianas/química , Ribosomas/química , Adenosina/química , Cristalografía por Rayos X , Código Genético , Rayos Láser , ARN Mensajero/química , ARN Mensajero/genética , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Ribosomas/genética , Temperatura , Thermus thermophilus/química , Rayos XRESUMEN
PCR clamping by locked nucleic acid (LNA) oligonucleotides is an effective technique for selectively amplifying the community SSU rRNA genes of plant-associated bacteria. However, the original primer set often shows low amplification efficiency. In order to improve this efficiency, new primers were designed at positions to compete with LNA oligonucleotides. Three new sets displayed higher amplification efficiencies than the original; however, efficiency varied among the primer sets. Two new sets appeared to be available in consideration of bacterial profiles by next-generation sequencing. One new set, KU63f and KU1494r, may be applicable to the selective gene amplification of plant-associated bacteria.