RESUMO
The correct folding of proteins is of paramount importance for their function, and protein misfolding is believed to be the primary cause of a wide range of diseases. Protein folding has been investigated with time-averaged methods and time-resolved spectroscopy, but observing the structural dynamics of the unfolding process in real-time is challenging. Here, we demonstrate an approach to directly reveal the structural changes in the unfolding reaction. We use nano- to millisecond time-resolved x-ray solution scattering to probe the unfolding of apomyoglobin. The unfolding reaction was triggered using a temperature jump, which was induced by a nanosecond laser pulse. We demonstrate a new strategy to interpret time-resolved x-ray solution scattering data, which evaluates ensembles of structures obtained from molecular dynamics simulations. We find that apomyoglobin passes three states when unfolding, which we characterize as native, molten globule, and unfolded. The molten globule dominates the population under the conditions investigated herein, whereas native and unfolded structures primarily contribute before the laser jump and 30 µs after it, respectively. The molten globule retains much of the native structure but shows a dynamic pattern of inter-residue contacts. Our study demonstrates a new strategy to directly observe structural changes over the cause of the unfolding reaction, providing time- and spatially resolved atomic details of the folding mechanism of globular proteins.
RESUMO
The electromagnetic polarizabilities of the nucleon are fundamental properties that describe its response to external electric and magnetic fields. They can be extracted from Compton-scattering data-and have been, with good accuracy, in the case of the proton. In contradistinction, information for the neutron requires the use of Compton scattering from nuclear targets. Here, we report a new measurement of elastic photon scattering from deuterium using quasimonoenergetic tagged photons at the MAX IV Laboratory in Lund, Sweden. These first new data in more than a decade effectively double the world data set. Their energy range overlaps with previous experiments and extends it by 20 MeV to higher energies. An analysis using chiral effective field theory with dynamical Δ(1232) degrees of freedom shows the data are consistent with and within the world data set. After demonstrating that the fit is consistent with the Baldin sum rule, extracting values for the isoscalar nucleon polarizabilities, and combining them with a recent result for the proton, we obtain the neutron polarizabilities as αn=[11.55±1.25(stat)±0.2(BSR)±0.8(th)]×10(-4) fm(3) and ßn=[3.65∓1.25(stat)±0.2(BSR)∓0.8(th)]×10(-4) fm(3), with χ(2)=45.2 for 44 degrees of freedom.
RESUMO
Photoneutron production on the nuclei of high-Z components of medical accelerator heads can lead to a significant secondary dose during a course of bremsstrahlung radiotherapy. However, a quantitative evaluation of secondary neutron dose requires improved data on the photoreaction yields. These have been measured as a function of photon energy, neutron energy and neutron angle for natW, using tagged photons at the MAX-Lab photonuclear facility in Sweden. This work presents neutron yields for natW(gamma, n) and compares these with the predictions of the Monte Carlo code MCNP-GN, developed specifically to simulate photoneutron production at medical accelerators.
Assuntos
Nêutrons , Fótons , Radioterapia de Alta Energia , Tungstênio/efeitos da radiação , Método de Monte Carlo , Dosagem Radioterapêutica , Radioterapia de Alta Energia/métodos , SoftwareRESUMO
Differential cross sections for Compton scattering from the deuteron were measured at MAX-Lab for incident photon energies of 55 and 66 MeV at nominal laboratory angles of 45 degrees, 125 degrees, and 135 degrees. Tagged photons were scattered from liquid deuterium and detected in three NaI spectrometers. By comparing the data with theoretical calculations in the framework of a one-boson-exchange potential model, the sum and the difference of the isospin-averaged nucleon polarizabilities, alpha(N)+beta(N)=17.4+/-3.7 and alpha(N)-beta(N)=6.4+/-2.4 (in units of 10(-4) fm(3)), have been determined. By combining the latter with the global-averaged value for alpha(p)-beta(p) and using the predictions of the Baldin sum rule for the sum of the nucleon polarizabilities, we have obtained values for the neutron electric and magnetic polarizabilities of alpha(n)=8.8+/-2.4(total)+/-3.0(model) and beta(n)=6.5-/+2.4(total)-/+3.0(model), respectively.
RESUMO
The purine-rich Shine-Dalgarno (SD) sequence located a few bases upstream of the mRNA initiation codon supports translation initiation by complementary binding to the anti-SD in the 16S rRNA, close to its 3' end. AUG is the canonical initiation codon but the weaker UUG and GUG codons are also used for a minority of genes. The codon sequence of the downstream region (DR), including the +2 codon immediately following the initiation codon, is also important for initiation efficiency. We have studied the interplay between these three initiation determinants on gene expression in growing Escherichia coli. One optimal SD sequence (SD(+)) and one lacking any apparent complementarity to the anti-SD in 16S rRNA (SD(-)) were analyzed. The SD(+) and DR sequences affected initiation in a synergistic manner and large differences in the effects were found. The gene expression level associated with the most efficient of these DRs together with SD(-) was comparable to that of other DRs together with SD(+). The otherwise weak initiation codon UUG, but not GUG, was comparable with AUG in strength, if placed in the context of two of the DRs. The +2 codon was one, but not the only, determinant for this unexpectedly high efficiency of UUG.
Assuntos
Códon de Iniciação/genética , DNA Bacteriano/genética , Biossíntese de Proteínas/genética , Sequência de Bases , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Óperon Lac/genética , Conformação de Ácido Nucleico , Plasmídeos/genética , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The genes for ribosomal proteins S4, S13 or S15 were fused with the gene for staphylococcal protein A, or derivatives thereof (2A'-7A'). The gene fusions were introduced into Escherichia coli strains, mutated in the corresponding ribosomal protein gene, by transformation. These mutated ribosomal proteins cause a phenotype that can be complemented. Thus, the phenotype of the transformants was tested and the ribosomal proteins were analyzed. The S4 N-terminal fusion protein severely disturbed growth of both the mutant and the wild-type strains. The S13 C-terminal fusion protein was proteolyzed close to the fusion point, giving a ribosomal protein moiety that could assemble into the ribosome normally. S15 N-terminal fusion proteins complemented a cold-sensitive strain lacking protein S15 in its ribosomes. These fused proteins were assembled into active ribosomes. The position of S15 in the 30S ribosomal subunit is well known. Therefore, in structural studies of the ribosome in vivo, the S15 fusion proteins can be used as a physical reporter for S15.
Assuntos
Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Sequência de Bases , Cromatografia Líquida , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteína Estafilocócica A/genéticaRESUMO
The codon that follows the AUG initiation triplet (+2 codon) affects gene expression in Escherichia coli. We have extended this analysis using two model genes lacking any apparent Shine-Dalgarno sequence. Depending on the identity of the +2 codon a difference in gene expression up to 20-fold could be obtained. The effects did not correlate with the levels of intracellular pools of cognate tRNA for the +2 codon, with putative secondary mRNA structures, or with mRNA stability. However, most +2 iso-codons that were decoded by the same species of tRNA gave pairwise similar effects, suggesting that the effect on gene expression was associated with the decoding tRNA. High adenine content of the +2 codon was associated with high gene expression. Of the fourteen +2 codons that mediated the highest efficiency, all except two had an adenine as the first base of the codon. Analysis of the 3540 E. coli genes from the TransTerm database revealed that codons associated with high gene expression in the two expression systems are over-represented at the +2 position in natural genes. Codons that are associated with low gene expression are under-represented. The data suggest that evolution has favored codons at the +2 position that give high translation initiation.
Assuntos
Códon de Iniciação/genética , Códon/genética , Escherichia coli/genética , Biossíntese de Proteínas , DNA Bacteriano/genética , DNA Recombinante , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Conformação de Ácido Nucleico , Plasmídeos/genética , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição GênicaRESUMO
OBJECTIVE: To find out whether we could identify prognostic factors for early failure of bypass to the foot in diabetic patients with critical ischaemia. DESIGN: Retrospective series of consecutive patients. SETTING: County hospital, Sweden. PATIENTS: 43 diabetic patients who had 48 reconstructions for critical ischaemia between 1988 and 1994. INTERVENTIONS: 48 elective vein bypass procedures to the feet. MAIN OUTCOME MEASURES: Prognostic factors for primary patency. RESULTS: Primary and secondary patency rates at one year were 72% (95% confidence interval (CI) 58 to 85) and 83% (95% CI 71 to 95), respectively. Limb salvage and survival rates at one year were 85% (95% CI 74 to 96) and 86% (95% CI 75 to 96), respectively. Vein graft of questionable quality, major wound healing problems, use of the reversed vein technique, and a narrow lumen (< 1.5 mm) of the recipient artery increased the hazard for failed primary patency by 17.3 (p = 0.003), 6.0 (p = 0.02), 4.7 (p = 0.03), and 3.9 (p = 0.05) times, respectively. Short vein bypass (p = 0.70), translocated or composite veins (p = 0.61), major postoperative oedema of the leg (p = 0.46), or questionable quality of the wall of the recipient artery (p = 0.29), however, had no significant independent effect on the primary patency rate. CONCLUSION: Early primary patency after bypass to the foot in diabetic patients might improve if veins of questionable quality, major wound healing problems, thin reversed veins from the calf, and narrow recipient arteries can be avoided or handled more proficiently than in the present study.
Assuntos
Derivação Arteriovenosa Cirúrgica , Angiopatias Diabéticas/cirurgia , Isquemia/cirurgia , Perna (Membro)/irrigação sanguínea , Perna (Membro)/cirurgia , Grau de Desobstrução Vascular , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Fatores de Risco , Fatores de TempoRESUMO
Termination efficiency of a mutant form of RF (release facor) 1, as compared to the wild-type enzyme, is influenced by the P-site peptidyl-tRNA if the termination signal is UAGA. This effect is weaker at the stronger termination signal UAGU. Similarly, low efficiency of the mutant RF1, together with certain peptidyl-tRNAs, can be increased by changing the second base of the 3'-flanking codon from C to G. The data suggest that the mutant RF1 interacts with the P-site peptidyl-tRNA in conjunction with the context at the 3'-side of the termination codon.
Assuntos
Códon de Terminação/genética , Proteínas de Escherichia coli , Mutação , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Códon de Terminação/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Terminação Traducional da Cadeia Peptídica , Ribossomos/metabolismoRESUMO
The outcome of out-of-hospital cardiac arrest (CA) following cardiopulmonary resuscitation (CPR) initiated by dispatcher-provided telephone instructions (T-CPR) in the area of Gothenburg, Sweden was studied. During a period of 27 months, 475 cases categorized by the dispatchers at the Emergency Co-ordination and Dispatch Centre as being suspected CA were offered T-CPR and were included in one of the following groups: (1) T-CPR completed (caller without previous CPR training); (2) T-CPR completed (caller with previous CPR training); (3) T-CPR started, but not completed; (4) T-CPR declined by caller due to previous CPR training; (5) T-CPR declined by caller due to other reasons; or, (6) T-CPR not offered. Of the patients, 473 could be followed up and of them 427 fulfilled the criteria for CA on ambulance arrival. Among the latter cases, 10% were hospitalized alive, 4% could be discharged from hospital, and the distribution among groups was: (1) 7%; (2) 18%; (3) 5%; (4) 11%; (5) 3%; and (6) 1%. The study concludes that although more attention should be paid to the detection of CA patients by the dispatchers, when the dispatchers suspected CA, their accuracy was high. Half of the witnesses accepted the offer of T-CPR and one-third completed T-CPR. More efforts and research are needed, however, to increase the percentages of callers completing CPR. The impact of T-CPR on survival might be limited. Indeed, the comparison of 'resuscitable' patients in whom T-CPR played an important role in supporting bystanders (i.e. groups 1 and 2) with 'resuscitable' patients in whom T-CPR was not performed (i.e. groups 3, 5 and 6) suggests an increase in survival from 6% (groups 3, 5 and 6) to 9% (groups 1 and 2).
Assuntos
Reanimação Cardiopulmonar/educação , Serviços Médicos de Emergência , Parada Cardíaca/terapia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Parada Cardíaca/mortalidade , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Taxa de SobrevidaRESUMO
Translation termination in vivo was studied in the yeast Saccharomyces cerevisiae using a translation-assay system. Codon changes that were made at position -2 relative to the stop codon, gave a 3.5-fold effect on termination in a release-factor-defective (sup45) mutant strain, in line with the effect observed in a wild-type strain. The influence of the -2 codon could be correlated to the charge of the corresponding amino acid residue in the nascent peptide; an acidic residue favoring efficient termination. Thus, the C-terminal end of the nascent peptide influences translation termination both in the bacterium Escherichia coli and to a lesser extent in the yeast S. cerevisiae. However, the sensitivity to the charge of the penultimate amino acid is reversed when the E. coli and S. cerevisiae are compared. Changing - 1 (P-site) codons in yeast gave a 10-fold difference in effect on the efficiency of termination. This effect could not be related to any property of the encoded last amino acid in the nascent peptide. Iso-codons read by the same tRNA (AAA/G, GAA/G) gave similar readthrough values. Codons for glutamine (CAA/G), glutamic acid (GAA/G) and isoleucine (AUA/C) that are read by different isoaccepting tRNAs are associated with an approximately twofold difference in each case in termination efficiency. This suggests that the P-site tRNA is able to influence termination at UGAC in yeast.
Assuntos
Códon de Terminação , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Sequência de Bases , Escherichia coli/genética , RNA Fúngico/genética , RNA de Transferência/genética , Proteína Estafilocócica A/genéticaRESUMO
The last two amino acids in the nascent peptide influence translation termination in E. coli (Mottagui-Tabar et al., 1994; Björnsson et al., 1996). We have compared the effects on termination in Escherichia coli, Bacillus subtilis and Salmonella typhimurium obtained by varying the -1 and -2 codons upstream of the weak UGAA stop signal. The peptide effect from the penultimate amino acid on translation termination in B. subtilis is similar to that seen in E. coli (with 66.5% RF-2 amino acid sequence similarity), whereas the influence in S. typhimurium (with 95.3% similarity to E. coli) is weaker. The effect of changing the -1 codon (P-site) is weaker in S. typhimurium as compared to those in E. coli and B. subtilis. RF-2s from E. coli and S. typhimurium have a threonine or alanine at position 246, respectively. This amino acid exchange in RF-2 can explain the difference in efficiency and toxicity during overexpression when E. coli and S. typhimurium are compared (Uno et al., 1996). However, B. subtilis RF-2 also has an alanine at that position, yet the sensitivity to the nascent peptide is similar to that in E. coli. Thus, the amino acid difference at position 246 in the RF-2 sequences cannot explain why termination in E. coli and B. subtilis is similar in peptide sensitivity while being different from that in S. typhimurium. Sequence alignments of RF-2 from the three bacteria show other regions of the molecule that could be involved in the functional interactions with the C-terminal end of the nascent peptide.
Assuntos
Bacillus subtilis/genética , Códon de Terminação/genética , Escherichia coli/genética , Iniciação Traducional da Cadeia Peptídica/genética , Terminação Traducional da Cadeia Peptídica/genética , Salmonella typhimurium/genética , Sequência de Aminoácidos , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post-termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature-sensitive Escherichia coli mutants which we isolated in this study. At non-permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re-initiate translation at various sites in all frames without the presence of an initiation codon. Re-initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas , Ribossomos/metabolismo , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Cromossomos Bacterianos/genética , Códon/fisiologia , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Dados de Sequência Molecular , Mutagênese , Iniciação Traducional da Cadeia Peptídica/genética , Terminação Traducional da Cadeia Peptídica/genética , Fenótipo , Proteínas Ribossômicas , TemperaturaRESUMO
Computer-aided electron tomography has been used to visualize ribosomes in Escherichia coli cells treated with kirromycin. This antibiotic stops bacterial growth by blocking the release of EF-Tu. GDP from the ribosome after GTP cleavage. Ribosomes in the kirromycin-treated cells are very compact, with the two subunits in close contact with each other. This closed structure is different from the open structure with spatially separated subunits that characterizes ribosomes in tryptophan-starved cells, giving deficiency for tryptophanyl.tRNA. A comparison of ribosomes in exponentially growing bacteria suggests that most ribosomes in an undefined working mode are in the closed conformation.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Ribossomos/ultraestrutura , Triptofano/fisiologia , Escherichia coli/ultraestrutura , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica/métodos , Piridonas/farmacologia , Ribossomos/químicaRESUMO
Ribosomes have different conformations in cells that are starved for a required amino acid (giving aminoacyl.tRNA starvation), or treated with kirromycin (blocking EF-Tu.GDP release), or are in exponential growth. A tunnel spans the 50S ribosome from a location facing the 70S ribosomal intersubunit space to the back side of the subunit in Escherichia coli cells. Here we have analyzed the internal low density region that corresponds to this tunnel in ribosomes in vivo. The data suggest that the tunnel is opened in connection with spatial separation of the subunits in ribosomes that have an empty A-site due to starvation for aminoacyl.tRNA. A region that corresponds to this tunnel can be found in the more compact structure of ribosomes in kirromycin-treated cells only after a substantial removal of low density material. This region is even less prominent in ribosomes in undefined working modes in growing bacteria. The data suggest that appearance of the tunnel through the 50S ribosomal subunit is working-mode dependent and it is not a characteristic feature of the major fraction of the ribosomal population in growing cells.
Assuntos
Escherichia coli/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Ribossomos/ultraestrutura , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Microscopia Eletrônica/métodos , Piridonas/farmacologia , Triptofano/fisiologiaRESUMO
Nonsense codon readthrough and changed translational reading frame were measured in different growth phases in E. coli. The strains used carry plasmid constructs with a translation assay reporter gene. This reporter gene contains an internal stop codon or a run of U-residues. Termination or frameshifting give rise to stable proteins that can be physically quantified on gels along with the complete protein products. Readthrough of the stop codon UGA by a nearcognate tRNA is several fold higher in active growth than in late exponential phase. In early exponential phase, about 7% of -1 frameshift at a U9 slippery sequence is detectable; upon entry to stationary phase this frameshifting increases to about 40% followed by a decrease in stationary phase. A similar increase is observed in the case of +1 reading frameshift at the U9 sequence, which increases from 13% in early exponential growth phase up to 38% at the beginning of stationary phase followed by a decrease. Thus, the levels of both stop codon readthrough and frameshifting are growth phase dependent, though not in an identical fashion.
Assuntos
Códon de Terminação , Escherichia coli/genética , Mudança da Fase de Leitura do Gene Ribossômico , Fases de Leitura , Sequência de Bases , DNA Bacteriano , Escherichia coli/crescimento & desenvolvimento , Dados de Sequência MolecularRESUMO
The glycine codons GGA or GGG, on the 5' side of stop codons UAG and UGA, are associated with a uniquely low termination efficiency in Escherichia coli, as compared to other codons, including the two glycine codons GGU and GGC. In contrast to the wild-type strain, all four glycine codons have a similar effect on termination at UAG in a strain with a mutant release factor 1 (RF1). Thus, these two glycine codon pairs, when present at the ribosomal P-site, affect termination efficiency of mutant or wild-type RF1 at UAG differently. If reading of GGA/G by tRNAGly2 is eliminated in the RF1 wild-type strain and replaced by a mutant form of tRNAGly3, termination efficiency is increased to the same level as for GGU/C, normally read by tRNAGly3. The results suggest an unusual interaction between the P-site tRNAGly2 and wild-type RF1 at the ribosomal A-site that is not present with mutant RF1.
Assuntos
Códon de Terminação/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Fatores de Terminação de Peptídeos/genética , RNA de Transferência de Glicina/metabolismo , Ribossomos/metabolismo , Elementos de DNA Transponíveis , Glicina/genética , Mutação , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Glicina/genética , Ribossomos/genéticaRESUMO
A cis -cleaving hammerhead ribozyme (Rz) expression system (3A'-Rz) in Escherichia coli has been constructed that can be used to study the involvement of factors that affect ribozyme cleavage in vivo . The ribozyme sequence is placed in the coding region of 3A' mRNA, which is expressed from a semi-synthetic translation assay gene. The size and the 5'-end sequences of the 3' cleavage fragments were determined and the efficiencies of different Rz variants were measured by quantitative primer extension. It is shown that one of the semi-active constructs (3A'-RzIII) can be used as an indicator for ribosomes that read through or terminate at a stop codon upstream of the Rz hammerhead sequence in the mRNA. Readthrough of the stop codon in an uncleaved mRNA gives a full length 3A' protein. Termination at the stop codon upstream of the ribozyme sequence gives a shortened termination product. However, the mRNA fragment that should arise as a result of the auto-cleavage does not give rise to any detectable corresponding truncated protein. Besides studies on translating ribosomes, the 3A'-Rz system can be used to isolate mutant strains that are changed in ribozyme activity either from internal base alterations, or changed interacting host factors.