RESUMEN
In all genomes, most amino acids are encoded by more than one codon. Synonymous codons can modulate protein production and folding, but the mechanism connecting codon usage to protein homeostasis is not known. Here we show that synonymous codon variants in the gene encoding gamma-B crystallin, a mammalian eye-lens protein, modulate the rates of translation and cotranslational folding of protein domains monitored in real time by Förster resonance energy transfer and fluorescence-intensity changes. Gamma-B crystallins produced from mRNAs with changed codon bias have the same amino acid sequence but attain different conformations, as indicated by altered in vivo stability and in vitro protease resistance. 2D NMR spectroscopic data suggest that structural differences are associated with different cysteine oxidation states of the purified proteins, providing a link between translation, folding, and the structures of isolated proteins. Thus, synonymous codons provide a secondary code for protein folding in the cell.
Asunto(s)
Pliegue de Proteína , Mutación Silenciosa , gamma-Cristalinas/biosíntesis , gamma-Cristalinas/genética , Secuencia de Aminoácidos , Clonación Molecular , Cisteína , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Genotipo , Cinética , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Péptido Hidrolasas/metabolismo , Fenotipo , Desnaturalización Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Solubilidad , gamma-Cristalinas/químicaRESUMEN
Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements. However, beyond differential subunit expression during development, evidence for regulated ribosome specification within individual cells has remained elusive. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5' untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders.
Asunto(s)
Mimetismo Biológico , Proteínas de Neoplasias/metabolismo , Biosíntesis de Proteínas , ARN Viral/metabolismo , Receptores de Cinasa C Activada/metabolismo , Ribosomas/metabolismo , Virus Vaccinia/enzimología , Proteínas Virales/metabolismo , Regiones no Traducidas 5'/genética , Adenosina/metabolismo , Secuencia de Aminoácidos , ARN Polimerasas Dirigidas por ADN/metabolismo , Humanos , Modelos Moleculares , Fosforilación , Poli A/metabolismo , ARN Viral/genética , Virus Vaccinia/genéticaRESUMEN
Receptor of activated protein C kinase 1 (RACK1) is a highly conserved eukaryotic protein that regulates several aspects of mRNA translation; yet, how it does so, remains poorly understood. Here we show that, although RACK1 consists largely of conserved ß-propeller domains that mediate binding to several other proteins, a short interconnecting loop between two of these blades varies across species to control distinct RACK1 functions during translation. Mutants and chimeras revealed that the amino acid composition of the loop is optimized to regulate interactions with eIF6, a eukaryotic initiation factor that controls 60S biogenesis and 80S ribosome assembly. Separately, phylogenetics revealed that, despite broad sequence divergence of the loop, there is striking conservation of negatively charged residues amongst protists and dicot plants, which is reintroduced to mammalian RACK1 by poxviruses through phosphorylation. Although both charged and uncharged loop mutants affect eIF6 interactions, only a negatively charged plant - but not uncharged yeast or human loop - enhances translation of mRNAs with adenosine-rich 5' untranslated regions (UTRs). Our findings reveal how sequence plasticity within the RACK1 loop confers multifunctionality in translational control across species.
Asunto(s)
Proteínas de Neoplasias/metabolismo , Unión Proteica , Receptores de Cinasa C Activada/metabolismo , Ribosomas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: Haemophilia B is caused by genetic aberrations in the F9 gene. The majority of these are non-synonymous mutations that alter the primary structure of blood coagulation factor IX (FIX). However, a synonymous mutation c.459G>A (Val107Val) was clinically reported to result in mild haemophilia B (FIX coagulant activity 15%-20% of normal). The F9 mRNA of these patients showed no skipping or retention of introns and/or change in mRNA levels, suggesting that mRNA integrity does not contribute to the origin of the disease in affected individuals. The aim of this study is to elucidate the molecular mechanisms that can explain disease manifestations in patients with this synonymous mutation. METHODS: We analyse the molecular mechanisms underlying the FIX deficiency through in silico analysis and reproducing the c.459G>A (Val107Val) mutation in stable cell lines. Conformation and non-conformation sensitive antibodies, limited trypsin digestion, activity assays for FIX, interaction with other proteins and post-translation modifications were used to evaluate the biophysical and biochemical consequences of the synonymous mutation. RESULTS: The Val107Val synonymous mutation in F9 was found to significantly diminish FIX expression. Our results suggest that this mutation slows FIX translation and affects its conformation resulting in decreased extracellular protein level. The altered conformation did not change the specific activity of the mutated protein. CONCLUSIONS: The pathogenic basis for one synonymous mutation (Val107Val) in the F9 gene associated with haemophilia B was determined. A mechanistic understanding of this synonymous variant yields potential for guiding and developing future therapeutic treatments.
Asunto(s)
Factor IX/química , Factor IX/genética , Hemofilia B/genética , Mutación Silenciosa/genética , Línea Celular Tumoral , Codón/genética , Factor IX/metabolismo , Factor VIIIa/química , Células HEK293 , Humanos , Proteínas Mutantes/metabolismo , Conformación Proteica , Procesamiento Proteico-Postraduccional , Estabilidad del ARN/genética , ARN Mensajero/química , ARN Mensajero/genética , TermodinámicaRESUMEN
Synonymous mutations, which do not alter the protein sequence, have been shown to affect protein function [Sauna ZE, Kimchi-Sarfaty C (2011) Nat Rev Genet 12(10):683-691]. However, synonymous mutations are rarely investigated in the cancer genomics field. We used whole-genome and -exome sequencing to identify somatic mutations in 29 melanoma samples. Validation of one synonymous somatic mutation in BCL2L12 in 285 samples identified 12 cases that harbored the recurrent F17F mutation. This mutation led to increased BCL2L12 mRNA and protein levels because of differential targeting of WT and mutant BCL2L12 by hsa-miR-671-5p. Protein made from mutant BCL2L12 transcript bound p53, inhibited UV-induced apoptosis more efficiently than WT BCL2L12, and reduced endogenous p53 target gene transcription. This report shows selection of a recurrent somatic synonymous mutation in cancer. Our data indicate that silent alterations have a role to play in human cancer, emphasizing the importance of their investigation in future cancer genome studies.
Asunto(s)
Apoptosis/genética , Regulación de la Expresión Génica/genética , Genoma Humano/genética , Melanoma/genética , Proteínas Musculares/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Secuencia de Bases , Western Blotting , Cartilla de ADN/genética , Exoma/genética , Vectores Genéticos/genética , Células HEK293 , Humanos , Inmunoprecipitación , Lentivirus , MicroARNs/genética , Datos de Secuencia Molecular , Proteínas Musculares/metabolismo , Mutación/genética , Polimorfismo de Nucleótido Simple/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
INTRODUCTION: Every woman has the right to respectful and empathetic care during childbirth that addresses her needs for pain management, and allows her the liberty to make it a memorable experience. This study aimed to assess the effect of birthing ball exercises on labor pain and labor outcome among primigravidae parturients at a tertiary care hospital. METHOD: A quasi-experimental design was used. A total of 60 primigravidae with 30 each in the control and experiment groups were selected by consecutive sampling. Primigravidae in the experiment group underwent two sessions of 20 minutes of birthing ball exercises at a subsequent gap of one hour during their active phase of labor (>4 cm cervical dilation). Primigravidae in the control group received routine standard care that included continuous observation and monitoring of vital signs and progress of labor. The visual analog scale (VAS) score was assessed in the transition phase (cervical dilation 8 cm to 10cm) and labor outcomes were assessed after delivery in both groups. RESULT: The experiment group had significantly better labor outcomes in terms of labor pain, cervical dilatation, and duration of labor compared to the primigravidae in the control group (p<0.05). In addition, the majority of mothers in the experiment group (86.7 %) underwent vaginal delivery with episiotomy compared to the control group (53.3%). Findings also revealed a statistically significant difference in the newborns of both groups regarding appearance, pulse, grimace, activity, and respiration (APGAR) score, crying immediately after birth, and admission to the neonatal intensive care unit (NICU) at p<0.05. CONCLUSION: There are a variety of discomforts that a woman experiences during labor. Reducing these discomforts is an important part of good nursing care. Non-pharmacologic methods like birthing ball exercises help decrease these discomforts by reducing labor pain and improving maternal and neonatal outcomes.
RESUMEN
The 9-aminoacridine (9AA) derivative quinacrine (QC) has a long history of safe human use as an antiprotozoal and antirheumatic agent. QC intercalates into DNA and RNA and can inhibit DNA replication, RNA transcription, and protein synthesis. The extent of QC intercalation into RNA depends on the complexity of its secondary and tertiary structure. Internal ribosome entry sites (IRESs) that are required for initiation of translation of some viral and cellular mRNAs typically have complex structures. Recent work has shown that some intercalating drugs, including QC, are capable of inhibiting hepatitis C virus IRES-mediated translation in a cell-free system. Here, we show that QC suppresses translation directed by the encephalomyocarditis virus (EMCV) and poliovirus IRESs in a cell-free system and in virus-infected HeLa cells. In contrast, IRESs present in the mammalian p53 transcript that are predicted to have less-complex structures were not sensitive to QC. Inhibition of IRES-mediated translation by QC correlated with the affinity of binding between QC and the particular IRES. Expression of viral capsid proteins, replication of viral RNAs, and production of virus were all strongly inhibited by QC (and 9AA). These results suggest that QC and similar intercalating drugs could potentially be used for treatment of viral infections.
Asunto(s)
Antivirales/farmacología , Virus de la Encefalomiocarditis/efectos de los fármacos , Poliovirus/efectos de los fármacos , Quinacrina/farmacología , Replicación Viral/efectos de los fármacos , Sitios de Unión , Virus de la Encefalomiocarditis/fisiología , Células HeLa , Humanos , Conformación de Ácido Nucleico , Poliovirus/fisiología , Biosíntesis de Proteínas/efectos de los fármacos , ARN Viral/metabolismo , Proteínas Virales/biosíntesisRESUMEN
Biosurfactant production was studied by Bacillus licheniformis K51, B. subtilis 20B, B. subtilis R1 and Bacillus strain HS3 using molasses or cheese whey as a sole source of nutrition at 45 degrees C. The isolates were able to grow and produce biosurfactant under shaking as well as static conditions. Maximum biosurfactant production was achieved with molasses at 5.0-7.0% (w/v). The biosurfactant retained its surface-active properties after incubation at 80 degrees C at a wide range of pH values and salt concentrations for nine days. Oil displacement experiments in sand pack columns with crude oil showed 25-33% recovery of residual oil.
Asunto(s)
Bacillus/metabolismo , Proteínas de la Leche/metabolismo , Melaza/microbiología , Tensoactivos/metabolismo , Eliminación de Residuos Líquidos/métodos , Bacillus/clasificación , Bacillus/crecimiento & desarrollo , Reactores Biológicos/microbiología , Técnicas de Cultivo de Célula/métodos , Concentración de Iones de Hidrógeno , Sales (Química)/metabolismo , Temperatura , Factores de Tiempo , Proteína de Suero de LecheRESUMEN
The possible application of a bacterial strain - Bacillus subtilis R1, isolated from an oil contaminated desert site in India, as biocontrol agent and its biosurfactant in microbial enhanced oil recovery are discussed. The biosurfactant production in minimal medium was carried out at different temperatures and salt concentrations, where it produced an efficient biosurfactant at 30-45°C and in presence of up to 7% salt. It significantly reduced the surface tension from 66±1.25mN/m to 29±0.85mN/m within 24h. In order to enhance the biosurfactant production, random mutagenesis of B. subtilis R1 was performed using chemical mutagen - ethyl methanesulfonate. Majority of the isolated 42 mutants showed biosurfactant production, but the difference was statistically insignificant as compared with parent strain R1. Therefore none of the mutants were selected for further study, and only parent strain R1 was studied. The biosurfactant was quite stable under harsh conditions for up to 10 days. The biosurfactant was extracted and characterized as similar to the lipopeptide group - surfactins and fengycin. The crude oil displacement experiments using biosurfactant broth in sand pack glass columns showed 33±1.25% additional oil recovery. The strain also showed inhibition of various plant pathogenic fungi on potato dextrose agar medium.
Asunto(s)
Bacillus subtilis/metabolismo , Lipopéptidos/biosíntesis , Antifúngicos/metabolismo , Antifúngicos/farmacología , Bacillus subtilis/clasificación , Bacillus subtilis/genética , Concentración de Iones de Hidrógeno , Lipopéptidos/farmacología , Ingeniería Metabólica , Pruebas de Sensibilidad Microbiana , Mutagénesis , ARN Ribosómico 16S/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectroscopía Infrarroja por Transformada de Fourier , Tensoactivos/metabolismo , Tensoactivos/farmacologíaRESUMEN
Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis. SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs. The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.
Asunto(s)
Proteínas de Escherichia coli/química , Péptidos/aislamiento & purificación , Ribosomas/química , Factores de Transcripción/química , gamma-Cristalinas/química , Secuencia de Aminoácidos , Animales , Bovinos , Proteínas de Escherichia coli/genética , Datos de Secuencia Molecular , Extensión de la Cadena Peptídica de Translación , Péptidos/genética , Péptidos/metabolismo , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Factores de Transcripción/genética , gamma-Cristalinas/genéticaRESUMEN
The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the message(for review see 1). However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates(1). Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others(2). Codon bias is organism as well as tissue specific(2,3). Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs(4). Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes(5-8). These pause sites can have functional impact on the protein expression, mRNA stability and protein folding(for review see 9). Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding(1,7,10,11). To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation. Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems(6-8). This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.
Asunto(s)
Péptidos/genética , Péptidos/aislamiento & purificación , Biosíntesis de Proteínas , ARN Mensajero/química , ARN Mensajero/genética , Ribosomas/química , Ribosomas/genética , Animales , Sistema Libre de Células , Centrifugación/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Extensión de la Cadena Peptídica de Translación , Conejos , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/metabolismoRESUMEN
The journey of nascent polypeptides from synthesis at the peptidyl transferase center of the ribosome ("birth") to full function ("maturity") involves multiple interactions, constraints, modifications and folding events. Each step of this journey impacts the ultimate expression level and functional capacity of the translated protein. It has become clear that the kinetics of protein translation is predominantly modulated by synonymous codon usage along the mRNA, and that this provides an active mechanism for coordinating the synthesis, maturation and folding of nascent polypeptides. Multiple quality control systems ensure that proteins achieve their native, functional form. Unproductive co-translational folding intermediates that arise during protein synthesis may undergo enhanced interaction with components of these systems, such as chaperones, and/or be subjects of co-translational degradation ("death"). This review provides an overview of our current understanding of the complex co-translational events that accompany the synthesis, maturation, folding and degradation of nascent polypeptide chains.
Asunto(s)
Péptidos , Biosíntesis de Proteínas/fisiología , Pliegue de Proteína , ARN Mensajero , Ribosomas/enzimología , Codón/metabolismo , Modelos Biológicos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Péptidos/genética , Péptidos/metabolismo , Peptidil Transferasas/genética , Peptidil Transferasas/metabolismo , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Ribosomas/genéticaRESUMEN
Abstract The possible application of a bacterial strain - Bacillus subtilis R1, isolated from an oil contaminated desert site in India, as biocontrol agent and its biosurfactant in microbial enhanced oil recovery are discussed. The biosurfactant production in minimal medium was carried out at different temperatures and salt concentrations, where it produced an efficient biosurfactant at 30-45 °C and in presence of up to 7% salt. It significantly reduced the surface tension from 66 ± 1.25 mN/m to 29 ± 0.85 mN/m within 24 h. In order to enhance the biosurfactant production, random mutagenesis of B. subtilis R1 was performed using chemical mutagen - ethyl methanesulfonate. Majority of the isolated 42 mutants showed biosurfactant production, but the difference was statistically insignificant as compared with parent strain R1. Therefore none of the mutants were selected for further study, and only parent strain R1 was studied. The biosurfactant was quite stable under harsh conditions for up to 10 days. The biosurfactant was extracted and characterized as similar to the lipopeptide group - surfactins and fengycin. The crude oil displacement experiments using biosurfactant broth in sand pack glass columns showed 33 ± 1.25% additional oil recovery. The strain also showed inhibition of various plant pathogenic fungi on potato dextrose agar medium.