Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered hydrolysis

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. (1) In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered hydrolysis

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. (1) In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

O uso de fosfatos associado à fitorremediação em solos de Santo Amaro (BA) contaminados por metais tóxicos

A contaminação do solo no município de Santo Amaro (BA) por metais tóxicos provocada pelas atividades da empresa Plumbum Mineração tem gerado impactos sobre a saúde ambiental e humana. O objetivo da presente pesquisa foi avaliar a viabilidade da remediação deste solo contaminado por chumbo e cádmio, utilizando diferentes fontes de fosfatos e fitorremediação com o capim vetiver [Vetiveria zizanioides (L.)]. O estudo foi realizado em colunas de PVC onde amostras de solo foram colocadas com a aplicação de fosfato dihidrogênio de potássio (KH2PO4) (T1); fertilizante fosfato natural reativo (FNR) (T2) e; uma mistura do KH2PO4 e de fertilizante FNR (T3). Amostras de solo contaminado sem tratamento (T0) foram utilizadas como controle. Após 60, 120 e 180 dias, alíquotas do solo foram retiradas das colunas para análises. Ao final de cada período, mudas de capim vetiver [(Vetiveria zizanioides (L.)] foram plantadas em vasos com as amostras de solo: T0, T1, T2 e T3 em triplicata. Para a determinação das concentrações de chumbo e cádmio no solo e tecidos vegetais foi utilizado o ICP-OES. A partir das análises física e química constatou-se que o solo possui textura argilosa e capacidade de troca catiônica (CTC) elevadas. As extrações com solução de ácido dietilenotriaminopentaacético (DTPA) e Toxicity Characteristic Leaching Procedure (TCLP) mostraram que o tratamento T1 seguido do T3 foram os mais eficientes na imobilização de Pb e Cd. Entretanto, todos os tratamentos resultaram em concentrações de metais ainda disponíveis no solo que excediam os limites estabelecidos pela USEPA, sendo o solo, portanto, considerado tóxico mesmo após o tratamento. Com base nas concentrações de metais extraídos através da extração sequencial pelo método BCR após a remediação e a fitorremediação do solo, foi verificado que todos os três tratamentos com fosfatos foram eficientes em imobilizar o Pb e Cd nas formas menos solúveis, porém, o Cd permaneceu mais solúvel e com maior mobilidade do que o Pb. Os ensaios de letalidade utilizando minhoca Eisenia andrei mostraram que a mortalidade observada no solo após 60 dias de tratamento foi significativamente reduzida após 120 e 180 dias de tratamento. A perda de biomassa pelas minhocas também foi reduzida de acordo com o tempo de tratamento. O teste de germinação com alfaces (Lactuca sativa L.) indicou que as amostras de solo tratadas continuam bastante tóxicas, apesar da disponibilidade reduzida do Pb e do Cd como visto nos resultados da extração por TCLP e por BCR. A avaliação de risco ecológico potencial indicou que os tratamentos do solo com fosfatos associado à fitorremediação reduziram a mobilidade do Pb, principalmente nos tratamentos T1 e T3. Para o Cd o risco ecológico potencial aumenta consideravelmente quando comparado com o Pb demonstrando que esse elemento, apesar dos tratamentos com fosfatos mais a fitorremediação continua móvel. O tratamento com KH2PO4 (T1) foi o mais eficiente na redução da mobilidade, disponibilidade e da toxicidade dos metais, seguido pelo T3 e T2 para o Pb e o T3 seguido pelo T1 e T2 para o Cd.

Phosphorus sources and fractions in an oxisol under no-tilled soybean

A dinâmica do P no solo pode ser modificada em sistemas com semeadura direta. Os agricultores brasileiros vêm aplicando fósforo na superfície do solo para otimizar a operação de máquinas, embora não seja uma prática recomendada pela pesquisa. Foi conduzido um experimento para estudar os efeitos da aplicação de duas fontes de fósforo na superfície do solo e no sulco de semeadura da soja. Os tratamentos consistiram da aplicação ou não de 80 kg ha-1 de P total como fosfato natural reativo e superfosfato, aplicados na superfície do solo, sobre os resíduos da cultura anterior. Na semeadura da soja, tratamentos adicionais (80 kg ha-1 de fosfato natural ou superfosfato) foram aplicados ao sulco de semeadura. O solo foi amostrado até 40 cm de profundidade, antes da semeadura da soja e após a colheita. Uma amostra controle foi tomada de área adjacente, sem cultivo. Houve aumento nos teores de P até 40 cm de profundidade, após a colheita da soja, de modo que o aumento foi observado principalmente no P ligado ao Ca e no P orgânico. Entretanto houve decréscimo nos teores de P ligado ao Fe. A adubação com ambas as fontes de fosfato levaram a um decréscimo nos teores de P ocluso na superfície do solo quando comparada com a área sem cultivo.

Variabilidade vertical de formas de fósforo em função de fontes e doses de fosfatos em semeadura direta

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Disponibilidade de fósforo para a soja cultivada sobre braquiária

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Substituição de fosfato solúvel por reativo em soja cultivada em semeadura direta em função de níveis de fósforo e modo de aplicação

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Corretivos de acidez e fontes de fósforo na disponibilidade de P no solo, nutrição e produção do amendoim e do capim marandu

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Efeito da compostagem sobre a solubilização e a eficiência agronômica de diferentes fontes de fósforo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Andropogon grass consortium with stylo in two times: the forage response and sources of phosphorus rates

The use of legumes intercropped with forage in the Brazilian savannah (Cerrado) region is an alternative to reverse the process of pasture degradation. The natural phosphorus deficiency in this region and the high retention capacity of this nutrient in the soil are factors that directly affect the sustainability of the production system. The objective of this study was to evaluate the effect of a phosphate with medium solubility (Arad) compared to more soluble sources such as triple superphosphate and mono-ammonium phosphate, on the formation and maintenance of pastures of Andropogon gayanus with or without the introduction of Stylosantes guianensis in pasture already established in the Brazilian savannah. Two experiments were carried out under field conditions in an Oxisol using a randomized block design with split plots, plots with two sources of phosphorus (Arad and super-triple or MAP) and subplots with different levels of phosphorus. The study showed that andropogon grass, even though characterised as forage requiring low fertilization, responded to increased fertilization, especially with the use of more soluble sources of P at planting. From the second year on, with the introduction of Stylosantes guianensis, verified effects were seen on legumes in their efficiency of utilization of residual fertilization from the first year; however, this didn't affect legume dry matter production resulting from the P sources used for the andropogon grass. The mixed stands of andropogon grass and Stylosantes guianensis made it more advantageous to increase the P sources using phosphate fertilizer with lower solubility (Arad), when compared to MAP.

Solubilización de fuentes de fósforo asociadas a un compuesto orgánico enriquecido con biofertilizantes

The enrichment of an organic compound with biofertilizers of microorganisms (solubilizing phosphorus bacteria, BSF) can improve the properties of phosphate fertilizers applied to soils to increase nutrient availability. The experimental design was randomized complete blocks with a factorial 4x2 +1 arrangement of treatments: 1) four sources of phosphorus: phosphate rock, natural phosphate, triple superphosphate (TSP) and Cuban phosphate rock; 2) two sources of bio-fertilizer prepared from filter cake (cane waste) enriched with microorganisms and no enrichment; 3) and an absolute control (no filter cake or fertilizer); repeated three times. Data was analyzed with ANOVA and treatment means were compared using Tukey (p <= 0.05). Assessments of available P (Bray-2 and Oniani methodology) and the quantification of total and solubilizing bacteria and fungi in the soil were performed 30, 60 and 90 d after starting the experiment. After 60 d, the population of BSF was lower in the control (p <= 0.05) and the content of P (Bray-2) was higher for SFT (p <= 0.01). The application of the bio-fertilizer increased the P content compared to the control. At 90 d P (Bray-2) increased with the SFT treatment, which also increased (p P in the soil at 60 and 90 d regardless of the presence of microorganism-enriched bio-fertilizer. The use of filter cake enriched with microorganisms associated with different sources of P, and applied to the soil with a high content of P, did not change the soil populations of total and solubilizing bacteria and fungi in the short term. TSP promoted the highest levels of P in the soil, irrespective of the presence of the organic compound enriched with P solubilizing microorganisms.

Arsenic removal from soil with high iron content using a natural surfactant and phosphate

An environment friendly arsenic removal technique from contaminated soil with high iron content has been studied. A natural surfactant extracted from soapnut fruit, phosphate solution and their mixture was used separately as extractants. The mixture was most effective in desorbing arsenic, attaining above 70 % efficiency in the pH range of 4–5. Desorption kinetics followed Elovich model. Micellar solubilization by soapnut and arsenic exchange mechanism by phosphate are the probable mechanisms behind arsenic desorption. Sequential extraction reveals that the mixed soapnut–phosphate system is effective in desorbing arsenic associated with amphoteric–Fe-oxide forms. No chemical change to the wash solutions was observed by Fourier transform-infrared spectra. Soil:solution ratio, surfactant and phosphate concentrations were found to affect the arsenic desorption process. Addition of phosphate boosted the performance of soapnut solution considerably. Response surface methodology approach predicted up to 80 % desorption of arsenic from soil when treated with a mixture of ≈1.5 % soapnut, ≈100 mM phosphate at a soil:solution ratio of 1:30.

Effect of ultrasonic instrumentation on the bond strength of crowns cemented with zinc phosphate cement to natural teeth. An in vitro study

Several studies have reported the benefits of sonic and/or ultrasonic instrumentation for root debridement, with most of them focusing on changes in periodontal clinical parameters. The present study investigated possible alterations in the tensile bond strength of crowns cemented with zinc phosphate cement to natural teeth after ultrasonic instrumentation. Forty recently extracted intact human third molars were selected, cleaned and stored in physiologic serum at 4°C. They received standard preparations, at a 16° convergence angle, and AgPd alloy crowns. The crowns were cemented with zinc phosphate cement and then divided into four groups of 10 teeth each. Each group was then subdivided into two subgroups, with one of the subgroups being submitted to 5,000 thermal cycles ranging from 55 ± 2 to 5 ± 2°C, while the other was not. Each group was submitted to ultrasonic instrumentation for different periods of time: group 1 - 0 min (control), group 2 - 5 min, group 3 - 10 min, and group 4 - 15 min. Tensile bond strength tests were performed with an Instron testing machine (model 4310). Statistical analysis was performed using ANOVA and Tukey's test at the 5% level of significance. A significant reduction in the tensile bond strength of crowns cemented with zinc phosphate and submitted to thermal cycles was observed at 15 min (196.75 N versus 0 min = 452.01 N, 5 min = 444.23 N and 10 min = 470.85 N). Thermal cycling and ultrasonic instrumentation for 15 min caused a significant reduction in tensile bond strength (p < .05).