Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes

Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon–anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the ‘GTP’- and ‘GDP’-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis.     

Genetic analysis, nucleotide sequence, and products of two Pseudomonas denitrificans cob genes encoding nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase and cobalamin (5''-phosphate) synthase.

Tn5 Sp(r) transposons have been inserted into the 8-kb Pseudomonas denitrificans DNA fragment from complementation group D, which carries cob genes. Genetic analysis and the nucleotide sequence revealed that only two cob genes (cobU and cobV) were found on this cob genomic locus. Nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase (EC 2.4.2.21) was assayed and purified to homogeneity from a P. denitrificans strain in which cobU and cobV were amplified. The purified enzyme was identified as the cobU gene product on the basis of identical molecular weights and N-terminal sequences. Cobalamin (5'-phosphate) synthase activity was increased when cobV was amplified in P. denitrificans. The partially purified enzyme catalyzed not only the synthesis of cobalamin 5'-phosphate from GDP-cobinamide and alpha-ribazole 5'-phosphate but also the one-step synthesis of cobalamin from GDP-cobinamide and alpha-ribazole. Biochemical data provided evidence that cobV encodes cobalamin (5'-phosphate) synthase.     

Identification and quantitation of corrinoid precursors of cobalamin from Pseudomonas denitrificans by high-performance liquid chromatography

After initial pretreatment for removal of interfering substances, corrinoid precursors of cobalamin from cultures of Pseudomonas denitrificans were separated by HPLC with a gradient elution system. In this system, all the following compounds are separated in their dicyano form, and retention times are given: cobyrinic acid; cobyrinic acid a-amide; cobyrinic acid c-amide; cobyrinic acid g-amide; cobyrinic acid a,g-diamide; cobyrinic acid c,g-diamide; cobyrinic acid a,c-diamide; cobyrinic acid a,c,g-triamide; cobyrinic acid triamide, tetraamide, and pentaamide isolated from P. denitrificans; cobyric acid; cobinamide; cobinamide phosphate; GDP-cobinamide; cyanocobalamin 5'-phosphate; and cyanocobalamin. Application of this HPLC method to culture samples of P. denitrificans revealed that in this microorganism the level of cobyrinic acid and cobyrinic acid monoamide is far lower than that of all other corrinoid precursors of cobalamin and suggested that (i) the (R)-1-amino-2-propanol group is incorporated only after completion of all the other amidations and (ii) the amidations follow only one sequence. The usefulness of this HPLC method was further demonstrated by identifying the 57Co-labeled corrinoid precursors of cobalamin accumulated by cobalamin-deficient mutants of Agrobacterium tumefaciens. A TLC system that separates the different corrinoid intermediates (in their dicyano form) and cyanocobalamin is also described.     

Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene.

Precorrin-6x reductase, which catalyzes the NADPH-dependent reduction of precorrin-6x to a dihydro derivative named precorrin-6y, was purified 14,300-fold to homogeneity with an 8% yield from extracts of a recombinant strain of Pseudomonas denitrificans. Precorrin-6y was identified by fast atom bombardment-mass spectrometry. It was converted in high yield (90%) to hydrogenobyrinic acid by cell-free protein preparations from P. denitrificans. For the purification and characterization of precorrin-6x reductase, a coupled-enzyme radioenzymatic assay was developed in which precorrin-6y was methylated in situ by the cobL gene product (F. Blanche, A. Famechon, D. Thibaut, L. Debussche, B. Cameron, J. Crouzet, J. Bacteriol. 174:1050-1052, 1992) in the presence of [methyl-3H]S-adenosyl-L-methionine. Molecular weights of precorrin-6x reductase obtained by gel filtration (Mr congruent to 27,000) and by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr congruent to 31,000) were consistent with the enzyme being a monomer. Km values of 3.6 +/- 0.2 microM for precorrin-6x and 23.5 +/- 3.5 microM for NADPH and a Vmax value of 17,000 U mg-1 were obtained at pH 7.7. The N-terminal sequence (six amino acids) and three internal sequences obtained after tryptic digestion of the enzyme were determined by microsequencing and established that precorrin-6x reductase is encoded by the cobK gene, located on a previously described 8.7-kb EcoRI fragment (J. Crouzet, B. Cameron, L. Cauchois, S. Rigault, M.-C. Rouyez, F. Blanche, D. Thibaut, and L. Debussche, J. Bacteriol. 172:5980-5990, 1990). However, the coding sequence was shown to be on the strand complementary to the one previously proposed as the coding strand.     

Assay, purification, and characterization of cobaltochelatase, a unique complex enzyme catalyzing cobalt insertion in hydrogenobyrinic acid a,c-diamide during coenzyme B12 biosynthesis in Pseudomonas denitrificans.

Hydrogenobyrinic acid a,c-diamide was shown to be the substrate of cobaltochelatase, an enzyme that catalyzes cobalt insertion in the corrin ring during the biosynthesis of coenzyme B12 in Pseudomonas denitrificans. Cobaltochelatase was demonstrated to be a complex enzyme composed of two different components of M(r) 140,000 and 450,000, which were purified to homogeneity. The 140,000-M(r) component was shown to be coded by cobN, whereas the 450,000-M(r) component was composed of two polypeptides specified by cobS and cobT. Each component was inactive by itself, but cobaltochelatase activity was reconstituted upon mixing CobN and CobST. The reaction was ATP dependent, and the Km values for hydrogenobyrinic acid a,c-diamide, Co2+, and ATP were 0.085 +/- 0.015, 4.2 +/- 0.2, and 220 +/- 36 microM, respectively. Spectroscopic data revealed that the reaction product was cob(II)yrinic acid a,c-diamide, and experiments with a coupled-enzyme incubation system containing both cobaltochelatase and cob(II)yrinic acid a,c-diamide reductase (F. Blanche, L. Maton, L. Debussche, and D. Thibaut, J. Bacteriol. 174:7452-7454, 1992) confirmed this result. This report not only provides the first evidence that hydrogenobyrinic acid and its a,c-diamide derivative are indeed precursors of adenosylcobalamin but also demonstrates that precorrin-6x, precorrin-6y, and precorrin-8x, three established precursors of hydrogenobyrinic acid (D. Thibaut, M. Couder, A. Famechon, L. Debussche, B. Cameron, J. Crouzet, and F. Blanche, J. Bacteriol. 174:1043-1049, 1992), are also on the pathway to cobalamin.     

Nucleotide sequence of a Pseudomonas denitrificans 5.4-kilobase DNA fragment containing five cob genes and identification of structural genes encoding S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase and cobyrinic acid a,c-diamide synthase.

A 5.4-kilobase DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment carries five different cob genes (cobA to cobE). Four of these genes present the characteristics of translationally coupled genes. cobA has been identified as the structural gene of S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) because the encoded protein has the same NH2 terminus and molecular weight as those determined for the purified SUMT. For the same reasons the cobB gene was shown to be the structural gene for cobyrinic acid a,c-diamide synthase. Genetic and biochemical data concerning cobC and cobD mutants suggest that the products of these genes are involved in the conversion of cobyric acid to cobinamide.     

Stopping or maintaining oral anticoagulation in patients undergoing photoselective vaporization of the prostate (SOAP) surgery for benign prostate obstruction: study protocol for a multicentre randomized controlled trial

Background

Lower urinary tract symptoms related to benign prostatic obstruction (BPO) are frequent in men aged >?50?years. Based on the use of innovative medical devices, a number of transurethral ablative techniques have recently been developed for the surgical treatment of BPO. In recent years, GreenLight photoselective vaporization of the prostate (PVP) has been considered as a non-inferior alternative to transurethral resection of the prostate. The GreenLight PVP is usually considered as an interesting surgical option for patients treated via oral anticoagulants (OACs) with regard to its haemostatic properties. The aim of this study was to assess the impact of maintaining OAC treatment in patients undergoing PVP.

Methods

This study is a multicentre, open-label, randomized controlled trial (RCT) designed to show the non-inferiority of PVP surgery in patients with BPO treated with OACs. This study is designed to enrol 386 OAC-treated patients (treated with vitamin K antagonists and direct oral anticoagulants) who are undergoing PVP for BPO. Patients will be randomized (1:1) to either maintain or stop OAC treatment during the perioperative course. The intervention group will maintain OAC treatment until the day before surgery and resume OAC treatment the day after surgery, whereas the control group will stop OAC treatment (with or without low-molecular-weight heparin bridging therapy) according to the anaesthesia guidelines. The primary outcome of interest to be assessed is the 30-day complications rate according to the Clavien-Dindo classification. The secondary endpoint will examine the 30-day rate of haemorrhagic and thrombotic events. This study will provide 80% power to show non-inferiority, defined as not worse than a 10% (non-inferiority margin) inferior change in the proportion of patients with good outcomes (Clavien-Dindo score?<?2), using two-tailed 95% confidence intervals.

Discussion

This first multicentre RCT in the field is underway to evaluate the safety and efficacy of PVP in patients with ongoing OAC therapy. The study results could influence the perioperative management of OACs in BPO surgery with a high level of evidence.

Trial registration

ClinicalTrials.gov, NCT03297281. Registered on 29 September 2017.

     

Identification and analysis of genes from Streptomyces pristinaespiralis encoding enzymes involved in the biosynthesis of the 4-dimethylamino-l-phenylalanine precursor of pristinamycin I

Four pap genes ( papA , papB , papC , papM  ) were found by sequencing near to snbA , a Streptomyces pristinaespiralis gene which was previously shown to encode one of the pristinamycin I (PI) synthetases. Analysis of the homologies observed from the deduced amino acid sequences suggested that these four genes could be involved in the biosynthesis of the PI precursor 4-dimethylamino- l -phenylalanine (DMPAPA). This was first verified when disruption of papA in S. pristinaespiralis led to a PI⁻ phenotype, which was reversed by the addition of DMPAPA into the culture medium. Further confirmation was obtained when papM was overexpressed in Escherichia coli and the corresponding protein purified to homogeneity. It catalysed the two successive N -methylation steps of 4-amino- l -phenylalanine leading to DMPAPA via 4-methylamino- l -phenylalanine. These results allowed us to assign a function to each of the four pap genes and to propose a biosynthetic pathway for DMPAPA.     

The highs and lows of dispersal: how connectivity and initial population size jointly shape establishment dynamics in discrete landscapes

Identifying the main factors driving introduced populations to establishment is a major challenge of invasion biology. Due to their small initial size, introduced populations are most vulnerable to extinction because of demographic stochasticity or Allee effects. While an increase in initial population size is known to increase establishment success, much remains to be understood regarding its interplay with connectivity in spatially structured environments. In order to better understand how demographic mechanisms interact at such spatial scale, we developed a stochastic model of population dynamics in discrete space to investigate the effect of connectivity and initial population size on establishment. The predictions derived from the model were then tested using experimental introductions of an insect parasitoid (Trichogramma chilonis) in spatially structured laboratory microcosms. Both theoretical and experimental results demonstrated that the connectivity of the introduction site had 1) a deleterious effect in the first generation when the introduced population was small and 2) a beneficial impact brought about by metapopulation effects in the subsequent generations. Interestingly, populations displayed a weakly pushed invasion pattern promoting early establishment, which was mainly underpinned by dispersal stochasticity and the discrete nature of the landscape. These results shed light on the critical influence of landscape connectivity on establishment dynamics.     

Tma108, a putative M1 aminopeptidase,is a specific nascent chain-associated protein in Saccharomyces cerevisiae

The discovery of novel specific ribosome-associated factors challenges the assumption that translation relies on standardized molecular machinery. In this work, we demonstrate that Tma108, an uncharacterized translation machinery-associated factor in yeast, defines a subpopulation of cellular ribosomes specifically involved in the translation of less than 200 mRNAs encoding proteins with ATP or Zinc binding domains. Using ribonucleoparticle dissociation experiments we established that Tma108 directly interacts with the nascent protein chain. Additionally, we have shown that translation of the first 35 amino acids of Asn1, one of the Tma108 targets, is necessary and sufficient to recruit Tma108, suggesting that it is loaded early during translation. Comparative genomic analyses, molecular modeling and directed mutagenesis point to Tma108 as an original M1 metallopeptidase, which uses its putative catalytic peptide-binding pocket to bind the N-terminus of its targets. The involvement of Tma108 in co-translational regulation is attested by a drastic change in the subcellular localization of ATP2 mRNA upon Tma108 inactivation. Tma108 is a unique example of a nascent chain-associated factor with high selectivity and its study illustrates the existence of other specific translation-associated factors besides RNA binding proteins.