Measurement of Compton scattering from the deuteron and an improved extraction of the neutron electromagnetic polarizabilities.

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.

Real-time tracking of protein unfolding with time-resolved x-ray solution scattering.

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.

Functional interactions between translation, transcription and ppGpp in growing Escherichia coli.

Strains with a relA mutation together with three different alleles of spoT were used to study the effects of different levels of ppGpp on production time for beta-galactosidase, transcriptional polarity and readthrough of a stop codon by near-cognate tRNA or a suppressor tRNA. The influences of an rpsL(S12) allele and a miaA mutation, together giving decreased efficiency of translation, as well as an rpoB mutation, coding for an altered RNA polymerase, were also investigated. The spoT alleles which give total deficiency for ppGpp, or a level which is increased several-fold (Sarubbi et al. (1988) Mol. Gen. Genet. 213, 214-222), had at the most a marginal effect on the production time for a beta-galactosidase molecule or translational misreading of a nonsense mutation. The efficiency of an amber tRNA suppressor is not affected by ppGpp in strains with an otherwise wildtype translational machinery. These data suggest that ppGpp does not influence directly the translational process in vivo. Instead, ppGpp is found to interfere with transcriptional readthrough in a manner which is dependent on the rpsL224, miaA, as well as the rpoB mutations. Similarly, bacterial growth is affected by ppGpp in a manner which is dependent on properties of both the transcriptional and translational apparatus together. It is suggested that the primary effect of ppGpp is on transcriptional readthrough, but this effect is modified by translational/transcriptional coupling.

Antisuppression by a mutation in rpsM(S13) giving a shortened ribosomal protein S13.

The phenotype associated with an rpsM(S13) mutation, originally isolated in Escherichia coli in a selection for pseudoreversion of streptomycin dependence, was studied in strains lacking the original mutations for antibiotic dependence. The rpsM mutation gives a decreased translational step time and a reduced growth rate. It functions as a strong antisuppressor to both the serU(Su1) amber suppressor and the trpT(Su9) opal suppressor, whereas the tyrT(Su3) amber suppressor is much less affected. The small ribosomal subunit from the rpsM mutant shows a reduced sedimentation coefficient but is able to form apparently normal 70S ribosomes as judged by ultracentrifugational analysis. Cloning and sequencing show that the rpsM mutation is a CAG to TAG alteration at codon position 100, giving an S13 protein which is shortened by 19 amino acids at its C-terminal end. This implies that the C-terminal domain of the protein that is involved in binding to 16S ribosomal RNA should be affected.

Ribosomes with large synthetic N-terminal extensions of protein S15 are active in vivo.

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.

UGA codon context which spans three codons. Reversal by ms2i6A37 in tRNA, mutation in rpsD(S4) or streptomycin.

Mutant UGA codon contexts which previously have been identified at different positions in the lacI part of a fused lacIlacZ gene were characterized with respect to translational readthrough in another genetic surrounding at a constant location. Although readthrough levels are systematically higher in this new location the "tight/leaky" characteristics of these codon contexts are essentially fully determined by the two codons flanking the nonsense codon itself. Analysis of some UGA hybrid contexts shows that the contribution to the codon context character by the codon either at the 5'-side (CCA or AGC) or at the 3'-side (NGU) is independent of the nature of the codon at the other side of UGA if this codon is decoded by trpT(Su9) suppressor tRNA. In a trpT(Su9), miaA double mutant strain, which lacks the ms2i6A37 modification in this tRNA, suppression is decreased at all UGA contexts investigated. However, in one case the contribution to the codon context character by the determinant flanking at one side is negatively affected by the nature of the codon at the other side of UGA. Thus, the character of a nonsense codon context in this case results from both flanking codons acting in a co-operative manner with the tRNA reading the middle UGA codon. This negative context effect is counteracted by a rpsD12 (ribosomal protein S4) mutation or by a sublethal concentration of streptomycin in the growth medium. It is suggested that the ms2i6A37 base in trpT(Su9) suppressor tRNA increases the efficiency of this tRNA by protecting it from ribosomal proofreading which is induced by codon context.

Functional interaction between tRNA2Gly2 at the ribosomal P-site and RF1 during termination at UAG.

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.

Functional interaction between release factor one and P-site peptidyl-tRNA on the ribosome.

Translation termination at UAG is influenced by the nature of the 5' flanking codon in Escherichia coli. Readthrough of the stop codon is always higher in a strain with mutant (prfA1) as compared to wild-type (prfA+) release factor one (RF1). Isocodons, which differ in the last base and are decoded by the same tRNA species, affect termination at UAG differently in strains with mutant or wild-type RF1. No general preference of the last codon base to favour readthrough or termination can be found. The data suggest that RF1 is sensitive to the nature of the wobble base anticodon-codon interaction at the ribosomal peptidyl-tRNA binding site (P-site). For some isoaccepting P-site tRNAs (tRNA3(Pro) versus tRNA2(Pro), tRNA4(Thr) versus tRNA1,3Thr) the effect is different on mutant and wild-type RF1, suggesting an interaction between RF1 at the aminoacyl-tRNA acceptor site (A-site) and the P-site tRNA itself. The glycine codons GGA (tRNA2(Gly)) and GGG (tRNA2,3(Gly)) at the ribosomal P-site are associated with an almost threefold higher readthrough of UAG than any of the other 42 codons tested, including the glycine codons GGU/C, in a strain with wild-type RF1. This differential response to the glycine codons is lost in the strain with the mutant form of RF1 since readthrough is increased to a similar high level for all four glycine codons. High alpha-helix propensity of the last amino acid residue at the C-terminal end of the nascent peptide is correlated with an increased termination at UAG. The effect is stronger on mutant compared to wild-type RF1. The data suggest that RF1-mediated termination at UAG is sensitive to the nature of the codon-anticodon interaction of the wobble base, the last amino acid residue of the nascent peptide chain, and the tRNA at the ribosomal P-site.

Genetic implication for an interaction between release factor one and ribosomal protein L7/L12 in vivo.

Strains with mutant alleles of the genes encoding release factor one (RF1), prfA, and ribosomal protein L7/L12, rplL, have been analyzed. The prfA1 allele has previously been shown to be associated with temperature sensitivity for growth at 43 degrees C and increased misreading of the nonsense codons UAG and UAA. Here we show that the prfA1 mutation is a nucleotide substitution that results in an arginine to proline change at amino acid position 137 in RF1. This alters the factor in a domain which may be involved in ribosome-binding. The rplL564 allele codes for a mutant form of protein L7/L12 that has a change in its conserved, translation-factor binding, domain. The rplL564 mutation suppresses the temperature sensitive phenotype and the increased translational misreading associated with the prfA1 allele, while other studied rplL mutant alleles do not. These data are consistent with an interaction between L7/L12 and RF1 in vivo.

The influence of the 5' codon context on translation termination in Bacillus subtilis and Escherichia coli is similar but different from Salmonella typhimurium.

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.

Cooperative effects by the initiation codon and its flanking regions on translation initiation.

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.

Codon bias at the 3'-side of the initiation codon is correlated with translation initiation efficiency in Escherichia coli.

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.

Only the last amino acids in the nascent peptide influence translation termination in Escherichia coli genes.

Efficiency of translation termination is affected if the last two amino acids in the nascent peptide are changed [1,2]. By changing the corresponding codons upstream of the stop signal UGAA, we have analyzed if the -3 to -6 amino acids at the C-terminal region of the nascent peptide also affect termination. Lysine at position -3 gave increased readthrough, whereas a total of 28 variations at positions -4, -5, and -6 showed no significant effect on readthrough. The 3'-ends corresponding to the last six codons in 27 Escherichia coli genes were inserted upstream of a stop codon in the 3A' translation assay gene [1]. Readthrough of the stop codon was measured and a possible correlation with the Codon Adaptation Index (CAI) 131 of the respective genes was investigated. Sequences from genes with low CAI do not give any such correlation, whereas sequences from genes with high CAI values are correlated with high termination efficiency. This correlation disappears if the -1 and -2 codons/amino acids are changed. The results suggest that mainly the terminal dipeptide of the terminal hexapeptide sequence has an influence on termination in the tested E. coli genes. This influence is dependent on the charge of the -2 amino acid and is correlated with the alpha-helix propensity of the -1 amino acid, in accordance with results obtained from synthetic gene constructs [1,2].

Interaction between a mutant release factor one and P-site peptidyl-tRNA is influenced by the identity of the two bases downstream of the stop codon UAG.

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.

Growth phase dependent stop codon readthrough and shift of translation reading frame in Escherichia coli.

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.

Contribution of normal and error-prone ribosomes to translational error formation in vivo.

Introduction of tRNA missense suppressors, and/or a protease deficiency into Escherichia coli strains has no significant effect on misreading of non-sense codons. An increased cellular level of faulty proteins therefore does not seem to have much secondary effect on translational accuracy. A genetic test system with two UGA non-sense mutations in the same fused lacIlacZ gene does not demonstrate any enrichment of error-prone ribosomes after read-through of the first non-sense codon in such strains. In contrast, the addition of sublethal amounts of streptomycin to a wild type strain appears to enrich error-prone ribosomes at the second non-sense codon, indicating the existence of a subpopulation of streptomycin-binding ribosomes. Ribosomes in a ribosomal ambiguity mutant strain (rpsD) with or without tRNA missense suppressors appear to be functionally homogeneous with respect to error production, as judged by read-through of the double UGA codons. The results that the major contribution to translational error formation in vivo originates from normal ribosomes and not from error-prone defective particles. An increased translational error in a bacterium results in very little, if any, increased functional heterogeneity of the ribosomal population with respect to error production. This suggests that an autocatalytic formation of translational errors is unlikely to occur in a growing bacterium.

Antagonistic effects of mutant elongation factor Tu and ribosomal protein S12 on control of translational accuracy, suppression and cellular growth.

Kirromycin-resistant mutant forms of elongation factor Tu, which are coded by tufA (Ar) or tufB (Bo) and are associated with an increased rate of translational error formation, have been analysed. In vivo, Ar was found to increase misreading as well as suppression of non-sense codons irrespective of Bo in a strain with wild type ribosomes. It is therefore not necessary to evoke both tufA (Ar) and tufB (Bo) mutations together in order to increase translational error as suggested earlier [1]. When combined with a hyperaccurate ribosomal rpsL (S12) mutation, Ar counteracts the restrictive effects on translational error formation caused by the altered protein S12, thus restoring the levels of missense error in vitro and non-sense error and suppression in vivo to near wild type values. As judged from in vitro experiments this results principally from a lowered selectivity of the Ar ternary complex at the initial discrimination step on the ribosome during translation. In vivo, this compensatory effect on the rpsL mutation on non-sense error formation and suppression is seen irrespective of the nature of tRNA or codon context. Furthermore, the tufA mutation enhances the cellular growth rate of the rpsL mutant, whereas it decreases growth of strains with normal ribosomes. Inactivation of one of the two genes coding for EF-Tu (tufB), while leaving the other gene (tufA) intact, can by itself, increase non-sense error formation and suppression.

Influence of the last amino acid in the nascent peptide on EF-Tu during decoding.

The last two amino acids of the nascent peptide at the ribosomal P-site influence the efficiency of termination readthrough at the stop codon UGA (Mottagui-Tabar et al (1994) EMBO J 13, 249-257; Björnsson et al (1996) EMBO J 15, 1696-1704). Here we analyze this effect on readthrough by wild type or a UGA suppressor form (Su9) of tRNA(Trp) by varying the codons at positions-1 and -2 at the 5' side of UGA. Strains with wild-type or mutant (ArBr) forms of elongation factor Tu (EF-Tu) were analyzed (Vijgenboom et al (1985) EMBO J4, 1049-1052). The effect on readthrough by changing these-1 and -2 codons is different on the two forms of tRNA(Trp) and is also dependent on the structure of EF-Tu. Readthrough by the tRNA(Trp)-derived suppressor, but not wild-type tRNA(Trp), is sensitive to the van der Waals volume of the last amino acid in the nascent peptide. Together with mutant EF-Tu, both forms of tRNA(Trp) are sensitive. The data suggest that the C-terminal amino acid in the nascent peptide is in a functional interaction with the EF-Tu ternary complex. This interaction is changed by mutation in tRNA(Trp) at position 24 or in EF-Tu at position 375. No indication of a changed interaction between the mutant EF-Tu and the penultimate amino acid could be found. Mutant forms of RF2 (Mikuni et al (1991) Biochimie 73, 1509-1516) and ribosomal proteins S4 and S12 (Fáxen et al (1988) J Bacteriol 170, 3756-3760) were found not be altered in sensitivity to the last two amino acids in the nascent peptide.

Photoneutron yields from tungsten in the energy range of the giant dipole resonance.

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.

Outcome for patients who call for an ambulance for chest pain in relation to the dispatcher's initial suspicion of acute myocardial infarction.

The very early handling of patients with suspected acute myocardial infarction (AMI) is of critical importance to the outcome. The aim of this study was to relate the dispatcher's initial suspicion of AMI, among patients who call for an ambulance due to chest pain, to the subsequent diagnosis and outcome. All patients who called for an ambulance in Gothenburg due to acute chest pain during a 2-month period were included in the study. In all, 503 patients fulfilled the inclusion criteria, and information on the dispatcher's initial suspicion of AMI was available in 484 patients. There was at least a strong suspicion of AMI in 36%, a moderate suspicion of AMI in 34% and only a vague or no suspicion in 30%. Among patients with at least a strong suspicion of AMI, 29% subsequently developed infarcation, compared with 18% among patients with a moderate suspicion of AMI and 15% among patients with only a vague or no suspicion (p < 0.001). However, the priority level was similar in patients with and without a life-threatening condition, and the mortality rate remained similar in patients with a strong suspicion and those without a strong suspicion of AMI. Thus, among patients who called for an ambulance due to acute chest pain there was a direct relationship between the dispatcher's suspicion of AMI and the subsequent diagnosis, but the mortality rate was similar in the different groups.