Roles of transcriptional and translational control mechanisms in regulation of ribosomal protein synthesis in Escherichia coli

ABSTRACTBacterial ribosome biogenesis is tightly regulated to match nutritional conditions and to prevent formation of defective ribosomal particles. InEscherichia coli, most ribosomal protein (r-protein) synthesis is coordinated with rRNA synthesis by a translational feedback mechanism: when r-proteins exceed rRNAs, specific r-proteins bind to their own mRNAs and inhibit expression of the operon. It was recently discovered that the second messenger nucleotide guanosine tetra and pentaphosphate (ppGpp), which directly regulates rRNA promoters, is also capable of regulating many r-protein promoters. To examine the relative contributions of the translational and transcriptional control mechanisms to the regulation of r-protein synthesis, we devised a reporter system that enabled us to genetically separate thecis-acting sequences responsible for the two mechanisms and to quantify their relative contributions to regulation under the same conditions. We show that the synthesis of r-proteins from the S20 and S10 operons is regulated by ppGpp following shifts in nutritional conditions, but most of the effect of ppGpp required the 5′ region of the r-protein mRNA containing the target site for translational feedback regulation and not the promoter. These results suggest that most regulation of the S20 and S10 operons by ppGpp following nutritional shifts is indirect and occurs in response to changes in rRNA synthesis. In contrast, we found that the promoters for the S20 operon were regulated during outgrowth, likely in response to increasing nucleoside triphosphate (NTP) levels. Thus, r-protein synthesis is dynamic, with different mechanisms acting at different times.IMPORTANCEBacterial cells have evolved complex and seemingly redundant strategies to regulate many high-energy-consuming processes. InE. coli, synthesis of ribosomal components is tightly regulated with respect to nutritional conditions by mechanisms that act at both the transcription and translation steps. In this work, we conclude that NTP and ppGpp concentrations can regulate synthesis of ribosomal proteins, but most of the effect of ppGpp is indirect as a consequence of translational feedback in response to changes in rRNA levels. Our results illustrate how effects of seemingly redundant regulatory mechanisms can be separated in time and that even when multiple mechanisms act concurrently their contributions are not necessarily equivalent.</jats:p

Enzyme-coupled assays for flip-flop of acyl-Coenzyme A in liposomes

Acyl-Coenzyme A is made in the cytosol. Certain enzymes using acyl-CoA seem to operate in the lumen of the ER but no corresponding flippases for acyl-CoA or an activated acyl have been described. In order to test the ability of purified candidate flippases to operate the transport of acyl-CoA through lipid bilayers in vitro we developed three enzyme-coupled assays using large unilamellar vesicles (LUVs) obtained by detergent removal. The first assay uses liposomes encapsulating a water-soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate (G3P). It measures formation of [3H]lyso-phosphatidic acid inside liposomes after [3H]palmitoyl-CoA has been added from outside. Two other tests use empty liposomes containing [3H]palmitoyl-CoA in the inner membrane leaflet, to which either soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate or alkaline phosphatase are added from outside. Here one can follow the appearance of [3H]lyso-phosphatidic acid or of dephosphorylated [3H]acyl-CoA, respectively, both being made outside the liposomes. Although the liposomes may retain small amounts of detergent, all these tests show that palmitoyl-CoA crosses the lipid bilayer only very slowly and that the lipid composition of liposomes barely affects the flip-flop rate. Thus, palmitoyl-CoA cannot cross the membrane spontaneously implying that in vivo some transport mechanism is required

Modified Differential Transform Method for Solving the Model of Pollution for a System of Lakes

This work presents the application of the differential transform method (DTM) to the model of pollution for a system of three lakes interconnected by channels. Three input models (periodic, exponentially decaying, and linear) are solved to show that DTM can provide analytical solutions of pollution model in convergent series form. In addition, we present the posttreatment of the power series solutions with the Laplace-Padé resummation method as a useful strategy to extend the domain of convergence of the approximate solutions. The Fehlberg fourth-fifth order Runge-Kutta method with degree four interpolant (RKF45) numerical solution of the lakes system problem is used as a reference to compare with the analytical approximations showing the high accuracy of the results. The main advantage of the proposed technique is that it is based on a few straightforward steps and does not generate secular terms or depend of a perturbation parameter

NGC 6309, a Planetary Nebula that Shifted from Round to Multipolar

We present new narrow-band Ha, [N II], and [O III] high-resolution images of the quadrupolar planetary nebula (PN) NGC 6309 that show in great detail its bipolar lobes and reveal new morphological features. New high- and low-dispersion long-slit spectra have been obtained to help in the investigation of the new nebular components. The images and spectra unveil two diffuse blobs, one of them located at 55 arcsec from the central star along the NE direction (PA= +71) and the other at 78 arcsec in the SW direction (PA= -151). Therefore, these structures do not share the symmetry axes of the inner bipolar outflows. Their radial velocities relative to the system are quite low: +3 and -4 km/s, respectively. Spectroscopic data confirm a high [O III] to Ha ratio, indicating that the blobs are being excited by the UV flux from the central star. Our images convincingly show a spherical halo 60 arcsec in diameter encircling the quadrupolar nebula. The expansion velocity of this shell is low, 66 km/s. The software SHAPE has been used to construct a morpho-kinematic model for the ring and the bipolar flows that implies an age of 4,000 yrs, the expansion of the halo sets a lower limit for its age 46,000 yrs, and the very low expansion of the blobs suggests they are part of a large structure corresponding to a mass ejection that took place 150,000 yrs ago. In NGC 6309 we have direct evidence of a change in the geometry of mass-loss, from spherical in the halo to axially-symmetric in the two pairs of bipolar lobes.Comment: 8 pages, 7 figures, Accepted for publication in MNRA

A Pediatrics-Based Instrument for Assessing Resident Education in Evidence-Based Practice

Objective:The principles of evidence-based practice (EBP) are a mandated component of the pediatric residency curriculum; however, a pediatrics-based assessment tool validated with pediatric residents does not exist. Methods: We designed an assessment instrument composed of items in 4 categories: 1) demographics; 2) comfort level; 3) self-reported practice of EBP; and 4) EBP knowledge. This last section required participants to identify best evidence and most appropriate study design by using pediatric-based scenarios, develop searchable questions, and use existing published research to address diagnostic and treatment issues. Four groups completed the instrument: preclinical medical students (MS-2), incoming pediatric interns (PGY-1), incoming second- and third-year pediatric residents (PGY2-3), and expert tutors (expert). We determined internal consistency, interrater reliability, content validity, item difficulty, and construct validity. Results: Fifty-six subjects completed tests (MS-2, n = 13; PGY-1, n = 13; PGY2-3, n = 22; expert, n = 8). Internal reliability was good, with Cronbach's alpha = .80. Interrater reliability was high (kappa = 0.94). Items were free of floor or ceiling effects. Comfort level and self-reported practice of EBP increased with expertise level and prior EBP experience (P < .01). Scores on the knowledge section (out of 50 +/- SD) rose with training level (MS-2: 14.8 +/- 5.7; PGY-1: 22.2 +/- 3.4; PGY2-3: 31.7 +/- 6.1; experts: 43 +/- 4.0; P < .01). Scores also correlated with prior EBP education. Conclusions: We have developed a reliable and valid instrument to assess knowledge and skill in EBP taught to pediatric residents. This instrument can aid pediatric educators in monitoring the impact of the EBP curriculum

Specification Design for an XML Mining Configurable Application

http://www.iaeng.org/publication/IMECS2011/IMECS2011_pp378-381.pdfInternational audienceThis work presents a methodology for XML mining centered on the process of extracting document features related to structure and content. This information is used to obtain similarity measures and to cluster XML documents. A conceptual framework is proposed to design an application with the primary goal of implementing a modular and easily configurable tool for mining large XML document collections

Analytical Solution of a Nonlinear Index-Three DAEs System Modelling a Slider-Crank Mechanism

The slider-crank mechanism (SCM) is one of the most important mechanisms in modern technology. It appears in most combustion engines including those of automobiles, trucks, and other small engines. The SCM model considered here is an index-three nonlinear system of differential-algebraic equations (DAEs), and therefore difficult to integrate numerically. In this work, we present the application of the differential transform method (DTM) to obtain an approximate analytical solution of the SCM model in convergent series form. In addition, we propose a posttreatment of the power series solution with the Padé resummation method to extend the domain of convergence of the approximate series solution. The main advantage of the proposed technique is that it does not require an index reduction and does not generate secular terms or depend on a perturbation parameter

Power Series Extender Method for the Solution of Nonlinear Differential Equations

We propose a power series extender method to obtain approximate solutions of nonlinear differential equations. In order to assess the benefits of this proposal, three nonlinear problems of different kind are solved and compared against the power series solution obtained using an approximative method. The problems are homogeneous Lane-Emden equation of α index, governing equation of a burning iron particle, and an explicit differential-algebraic equation related to battery model simulations. The results show that PSEM generates highly accurate handy approximations requiring only a few steps. The main advantage of PSEM is to extend the domain of convergence of the power series solutions of approximative methods as Taylor series method, homotopy perturbation method, homotopy analysis method, variational iteration method, differential transform method, and Adomian decomposition method, among many others. From the application of PSEM, it results in handy easy computable expressions that extend the domain of convergence of high order power series solutions

Chemogenetic E-MAP in Saccharomyces cerevisiae for identification of membrane transporters operating lipid flip flop

While most yeast enzymes for the biosynthesis of glycerophospholipids, sphingolipids and ergosterol are known, genes for several postulated transporters allowing the flopping of biosynthetic intermediates and newly made lipids from the cytosolic to the lumenal side of the membrane are still not identified. An E-MAP measuring the growth of 142'108 double mutants generated by systematically crossing 543 hypomorphic or deletion alleles in genes encoding multispan membrane proteins, both on media with or without an inhibitor of fatty acid synthesis, was generated. Flc proteins, represented by 4 homologous genes encoding presumed FAD or calcium transporters of the ER, have a severe depression of sphingolipid biosynthesis and elevated detergent sensitivity of the ER. FLC1, FLC2 and FLC3 are redundant in granting a common function, which remains essential even when the severe cell wall defect of flc mutants is compensated by osmotic support. Biochemical characterization of some other genetic interactions shows that Cst26 is the enzyme mainly responsible for the introduction of saturated very long chain fatty acids into phosphatidylinositol and that the GPI lipid remodelase Cwh43, responsible for introducing ceramides into GPI anchors having a C26:0 fatty acid in sn-2 of the glycerol moiety can also use lyso-GPI protein anchors and various base resistant lipids as substrates. Furthermore, we observe that adjacent deletions in several chromosomal regions show strong negative genetic interactions with a single gene on another chromosome suggesting the presence of undeclared suppressor mutations in certain chromosomal regions that need to be identified in order to yield meaningful E-map data

Model predictive control: a review of its applications in power electronics

Model-based predictive control (MPC) for power converters and drives is a control technique that has gained attention in the research community. The main reason for this is that although MPC presents high computational burden, it can easily handle multivariable case and system constraints and nonlinearities in a very intuitive way. Taking advantage of that, MPC has been successfully used for different applications such as an active front end (AFE), power converters connected to resistor inductor RL loads, uninterruptible power supplies, and high-performance drives for induction machines, among others. This article provides a review of the application of MPC in the power electronics area