Kinetic mechanism of elongation factor Ts-catalyzed nucleotide exchange in elongation factor Tu.

The interaction of Escherichia coli elongation factor Tu (EF-Tu) with elongation factor Ts (EF-Ts) and guanine nucleotides was studied by the stopped-flow technique, monitoring the fluorescence of tryptophan 184 in EF-Tu or of the mant group attached to the guanine nucleotide. Rate constants of all association and dissociation reactions among EF-Tu, EF-Ts, GDP, and GTP were determined. EF-Ts enhances the dissociation of GDP and GTP from EF-Tu by factors of 6 x 10(4) and 3 x 10(3), respectively. The loss of Mg(2+) alone, without EF-Ts, accounts for a 150-300-fold acceleration of GDP dissociation from EF-Tu.GDP, suggesting that the disruption of the Mg(2+) binding site alone does not explain the EF-Ts effect. Dissociation of EF-Ts from the ternary complexes with EF-Tu and GDP/GTP is 10(3)-10(4) times faster than from the binary complex EF-Tu.EF-Ts, indicating different structures and/or interactions of the factors in the binary and ternary complexes. Rate constants of EF-Ts binding to EF-Tu in the free or nucleotide-bound form or of GDP/GTP binding to the EF-Tu.EF-Ts complex range from 0.6 x 10(7) to 6 x 10(7) M(-1) s(-1). At in vivo concentrations of nucleotides and factors, the overall exchange rate, as calculated from the elemental rate constants, is 30 s(-1), which is compatible with the rate of protein synthesis in the cell.     

Designs for clinical trials with time-to-event outcomes based on stopping guidelines for lack of benefit

Background

We present the design, methods and population characteristics of a large community trial that assessed the efficacy of a weekly supplement containing vitamin A or beta-carotene, at recommended dietary levels, in reducing maternal mortality from early gestation through 12 weeks postpartum. We identify challenges faced and report solutions in implementing an intervention trial under low-resource, rural conditions, including the importance of population choice in promoting generalizability, maintaining rigorous data quality control to reduce inter- and intra- worker variation, and optimizing efficiencies in information and resources flow from and to the field.

Methods

This trial was a double-masked, cluster-randomized, dual intervention, placebo-controlled trial in a contiguous rural area of ~435 sq km with a population of ~650,000 in Gaibandha and Rangpur Districts of Northwestern Bangladesh. Approximately 120,000 married women of reproductive age underwent 5-weekly home surveillance, of whom ~60,000 were detected as pregnant, enrolled into the trial and gave birth to ~44,000 live-born infants. Upon enrollment, at ~ 9 weeks' gestation, pregnant women received a weekly oral supplement containing vitamin A (7000 ug retinol equivalents (RE)), beta-carotene (42 mg, or ~7000 ug RE) or a placebo through 12 weeks postpartum, according to prior randomized allocation of their cluster of residence. Systems described include enlistment and 5-weekly home surveillance for pregnancy based on menstrual history and urine testing, weekly supervised supplementation, periodic risk factor interviews, maternal and infant vital outcome monitoring, birth defect surveillance and clinical/biochemical substudies.

Results

The primary outcome was pregnancy-related mortality assessed for 3 months following parturition. Secondary outcomes included fetal loss due to miscarriage or stillbirth, infant mortality under three months of age, maternal obstetric and infectious morbidity, infant infectious morbidity, maternal and infant micronutrient status, fetal and infant growth and prematurity, external birth defects and postnatal infant growth to 3 months of age.

Conclusion

Aspects of study site selection and its "resonance" with national and rural qualities of Bangladesh, the trial's design, methods and allocation group comparability achieved by randomization, field procedures and innovative approaches to solving challenges in trial conduct are described and discussed. This trial is registered with http://Clinicaltrials.gov as protocol NCT00198822.     

Streptomycin interferes with conformational coupling between codon recognition and GTPase activation on the ribosome

Aminoacyl-tRNAs (aa-tRNAs) are selected by the ribosome through a kinetically controlled induced fit mechanism. Cognate codon recognition induces a conformational change in the decoding center and a domain closure of the 30S subunit. We studied how these global structural rearrangements are related to tRNA discrimination by using streptomycin to restrict the conformational flexibility of the 30S subunit. The antibiotic stabilized aa-tRNA on the ribosome both with a cognate and with a near-cognate codon in the A site. Streptomycin altered the rates of GTP hydrolysis by elongation factor Tu (EF-Tu) on cognate and near-cognate codons, resulting in almost identical rates of GTP hydrolysis and virtually complete loss of selectivity. These results indicate that movements within the 30S subunit at the streptomycin-binding site are essential for the coupling between base pair recognition and GTP hydrolysis, thus modulating the fidelity of aa-tRNA selection.     

Towards the development of a DNA-sequence based approach to serotyping of <Emphasis Type="Italic">Salmonella enterica</Emphasis>

Background  

The fliC and fljB genes in Salmonella code for the phase 1 (H1) and phase 2 (H2) flagellin respectively, the rfb cluster encodes the majority of enzymes for polysaccharide (O) antigen biosynthesis, together they determine the antigenic profile by which Salmonella are identified. Sequencing and characterisation of fliC was performed in the development of a molecular serotyping technique.     

Towards understanding selenocysteine incorporation into bacterial proteins

In bacteria, UGA stop codons can be recoded to direct the incorporation of selenocysteine into proteins on the ribosome. Recoding requires a selenocysteine incorporation sequence (SECIS) downstream of the UGA codon, a specialized translation factor SelB, and the non-canonical Sec-tRNASec, which is formed from Ser-tRNASec by selenocysteine synthase, SelA, using selenophosphate as selenium donor. Here we describe a rapid-kinetics approach to study the mechanism of selenocysteine insertion into proteins on the ribosome. Labeling of SelB, Sec-tRNASec and other components of the translational machinery allows direct observation of the formation or dissociation of complexes by monitoring changes in the fluorescence of single dyes or fluorescence resonance energy transfer between two fluorophores. Furthermore, the structure of SelA was studied by electron cryomicroscopy (cryo-EM). We report that intact SelA from the thermophilic bacterium Moorella thermoacetica (mthSelA) can be vitrified for cryo-EM using a controlled-environment vitrification system. Two-dimensional image analysis of vitrified mthSelA images shows that SelA can adopt the wide range of orientations required for high-resolution structure determination by cryo-EM. The results indicate that mthSelA forms a homodecamer that has a ring-like structure with five bilobed wings, similar to the structure of the E. coli complex determined previously.     

The ribosome's response to codon-anticodon mismatches

The ribosome is a molecular machine that synthesizes polypeptides from aminoacyl-tRNAs according to the sequence of the mRNA template. Codon reading by the anticodon of tRNA is controlled by a network of ribosome contacts that are specific for each position of the codon-anticodon duplex and involve A-minor RNA interactions. Rapid and accurate tRNA selection is accomplished by switching the conformation of the decoding site between accepting and rejecting mode, regardless of the thermodynamic stability of the respective codon-anticodon complexes or their interactions at the decoding site. The forward reactions are particularly sensitive to mismatches and determine the variations in the extent of misreading of near-cognate codons, both during initial selection and proofreading. This review emphasizes the progress made in understanding the mechanisms that determine recognition and selection of tRNA by the translational machinery.     

Structural and functional investigation of a putative archaeal selenocysteine synthase

Bacterial selenocysteine synthase converts seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec) for selenoprotein biosynthesis. The identity of this enzyme in archaea and eukaryotes is unknown. On the basis of sequence similarity, a conserved open reading frame has been annotated as a selenocysteine synthase gene in archaeal genomes. We have determined the crystal structure of the corresponding protein from Methanococcus jannaschii, MJ0158. The protein was found to be dimeric with a distinctive domain arrangement and an exposed active site, built from residues of the large domain of one protomer alone. The shape of the dimer is reminiscent of a substructure of the decameric Escherichia coli selenocysteine synthase seen in electron microscopic projections. However, biochemical analyses demonstrated that MJ0158 lacked affinity for E. coli seryl-tRNA(Sec) or M. jannaschii seryl-tRNA(Sec), and neither substrate was directly converted to selenocysteinyl-tRNA(Sec) by MJ0158 when supplied with selenophosphate. We then tested a hypothetical M. jannaschii O-phosphoseryl-tRNA(Sec) kinase and demonstrated that the enzyme converts seryl-tRNA(Sec) to O-phosphoseryl-tRNA(Sec) that could constitute an activated intermediate for selenocysteinyl-tRNA(Sec) production. MJ0158 also failed to convert O-phosphoseryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). In contrast, both archaeal and bacterial seryl-tRNA synthetases were able to charge both archaeal and bacterial tRNA(Sec) with serine, and E. coli selenocysteine synthase converted both types of seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). These findings demonstrate that a number of factors from the selenoprotein biosynthesis machineries are cross-reactive between the bacterial and the archaeal systems but that MJ0158 either does not encode a selenocysteine synthase or requires additional factors for activity.     

Recognition and selection of tRNA in translation

Aminoacyl-tRNA (aa-tRNA) is delivered to the ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. The stepwise movement of aa-tRNA from EF-Tu into the ribosomal A site entails a number of intermediates. The ribosome recognizes aa-tRNA through shape discrimination of the codon-anticodon duplex and regulates the rates of GTP hydrolysis by EF-Tu and aa-tRNA accommodation in the A site by an induced fit mechanism. Recent results of kinetic measurements, ribosome crystallography, single molecule FRET measurements, and cryo-electron microscopy suggest the mechanism of tRNA recognition and selection.     

Mechanism of EF-Ts-catalyzed guanine nucleotide exchange in EF-Tu: contribution of interactions mediated by helix B of EF-Tu

Elongation factor Tu (EF-Tu) belongs to the family of GTP-binding proteins and requires elongation factor Ts (EF-Ts) for nucleotide exchange. Crystal structures suggested that one of the salient features in the EF-Tu x EF-Ts complex is a conformation change in the switch II region of EF-Tu that is initiated by intrusion of Phe81 of EF-Ts between His84 and His118 of EF-Tu and may result in a destabilization of Mg2+ coordination and guanine nucleotide release. In the present paper, the contribution of His84 to nucleotide release was studied by pre-steady-state kinetic analysis of nucleotide exchange in mutant EF-Tu in which His84 was replaced by Ala. Both intrinsic and EF-Ts-catalyzed nucleotide release was affected by the mutation, resulting in a 10-fold faster spontaneous GDP release and a 4-fold faster EF-Ts-catalyzed release of GTP and GDP. Removal of Mg2+ from the EF-Tu x EF-Ts complex increased the rate constant of GDP release 2-fold, suggesting a small contribution to nucleotide exchange. Together with published data on the effects of mutations interfering with other putative interactions between EF-Tu and EF-Ts, the results suggest that each of the contacts in the EF-Tu x EF-Ts complex alone contributes moderately to nucleotide destabilization, but together they act synergistically to bring about the overall 60,000-fold acceleration of nucleotide exchange in EF-Tu by EF-Ts.