Variation in the organization and subunit composition of the mammalian pyruvate dehydrogenase complex E2/E3BP core assembly

The final version of this article is available at the link below.Crucial to glucose homoeostasis in humans, the hPDC (human pyruvate dehydrogenase complex) is a massive molecular machine comprising multiple copies of three distinct enzymes (E1–E3) and an accessory subunit, E3BP (E3-binding protein). Its icosahedral E2/E3BP 60-meric ‘core’ provides the central structural and mechanistic framework ensuring favourable E1 and E3 positioning and enzyme co-operativity. Current core models indicate either a 48E2+12E3BP or a 40E2+20E3BP subunit composition. In the present study, we demonstrate clear differences in subunit content and organization between the recombinant hPDC core (rhPDC; 40E2+20E3BP), generated under defined conditions where E3BP is produced in excess, and its native bovine (48E2+12E3BP) counterpart. The results of the present study provide a rational basis for resolving apparent differences between previous models, both obtained using rhE2/E3BP core assemblies where no account was taken of relative E2 and E3BP expression levels. Mathematical modelling predicts that an ‘average’ 48E2+12E3BP core arrangement allows maximum flexibility in assembly, while providing the appropriate balance of bound E1 and E3 enzymes for optimal catalytic efficiency and regulatory fine-tuning. We also show that the rhE2/E3BP and bovine E2/E3BP cores bind E3s with a 2:1 stoichiometry, and propose that mammalian PDC comprises a heterogeneous population of assemblies incorporating a network of E3 (and possibly E1) cross-bridges above the core surface.This work was partly supported by EPSRC (under grants GR/R99393/01 and EP/C015452/1)

Solution conformations of early intermediates in Mos1 transposition

DNA transposases facilitate genome rearrangements by moving DNA transposons around and between genomes by a cut-and-paste mechanism. DNA transposition proceeds in an ordered series of nucleoprotein complexes that coordinate pairing and cleavage of the transposon ends and integration of the cleaved ends at a new genomic site. Transposition is initiated by transposase recognition of the inverted repeat sequences marking each transposon end. Using a combination of solution scattering and biochemical techniques, we have determined the solution conformations and stoichiometries of DNA-free Mos1 transposase and of the transposase bound to a single transposon end. We show that Mos1 transposase is an elongated homodimer in the absence of DNA and that the N-terminal 55 residues, containing the first helix-turn-helix motif, are required for dimerization. This arrangement is remarkably different from the compact, crossed architecture of the dimer in the Mos1 paired-end complex (PEC). The transposase remains elongated when bound to a single-transposon end in a pre-cleavage complex, and the DNA is bound predominantly to one transposase monomer. We propose that a conformational change in the single-end complex, involving rotation of one half of the transposase along with binding of a second transposon end, could facilitate PEC assembly

MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island

Short-read, high-throughput sequencing technology cannot identify the chromosomal position of repetitive insertion sequences that typically flank horizontally acquired genes such as bacterial virulence genes and antibiotic resistance genes. The MinION nanopore sequencer can produce long sequencing reads on a device similar in size to a USB memory stick. Here we apply a MinION sequencer to resolve the structure and chromosomal insertion site of a composite antibiotic resistance island in Salmonella Typhi Haplotype 58. Nanopore sequencing data from a single 18-h run was used to create a scaffold for an assembly generated from short-read Illumina data. Our results demonstrate the potential of the MinION device in clinical laboratories to fully characterize the epidemic spread of bacterial pathogens

Cationic lipid : DNA complexes - their structure and interactions with model cell membranes

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Correction: Structural and Functional Analysis of the Symmetrical Type I Restriction Endonuclease R.EcoR124I(NT).

[This corrects the article DOI: 10.1371/journal.pone.0035263.]

Structural and Functional Analysis of the Symmetrical Type I Restriction Endonuclease R.EcoR124INT

Type I restriction-modification (RM) systems are comprised of two multi-subunit enzymes, the methyltransferase (,160 kDa), responsible for methylation of DNA, and the restriction endonuclease (,400 kDa), responsible for DNA cleavage. Both enzymes share a number of subunits. An engineered RM system, EcoR124INT, based on the N-terminal domain of the specificity subunit of EcoR124I was constructed that recognises the symmetrical sequence GAAN7TTC and is active as a methyltransferase. Here, we investigate the restriction endonuclease activity of R. EcoR124I NT in vitro and the subunit assembly of the multi-subunit enzyme. Finally, using small-angle neutron scattering and selective deuteration, we present a low-resolution structural model of the endonuclease and locate the motor subunits within the multi-subunit enzyme. We show that the covalent linkage between the two target recognition domains of the specificity subunit is not required for subunit assembly or enzyme activity, and discuss the implications for the evolution of Type I enzymes

A rich Ediacaran assemblage from eastern Avalonia : evidence of early widespread diversity in the deep ocean

The Avalon Assemblage (Ediacaran, late Neoproterozoic) provides some of the oldest evidence of diverse macroscopic life and underpins current understanding of the early evolution of epibenthic communities. However, its overall diversity and provincial variability are poorly constrained and are based largely on biotas preserved in Newfoundland, Canada. We report coeval high-diversity biotas from Charnwood Forest, UK, which share at least 60% of their genera in common with ones in Newfoundland. This indicates that substantial taxonomic exchange took place between different regions of Avalonia, probably facilitated by ocean currents, and suggests that a diverse deepwater biota may already have been widespread at the time. Contrasts in the relative abundance of prostrate versus erect taxa likely record differential sensitivity to physical environmental parameters (hydrodynamic regime, substrate) and highlight their significance in controlling community structure

Small-angle neutron scattering of R.EcoR124I_NT.

<p><b>(a)</b> the SANS profile of R.EcoR124I_NT measured in H₂O (blue triangles) and the SANS of the two HsdR subunits <i>in situ</i> from a sample containing deuterated HsdR subunits and protonated MTase, measured in 40% D₂O (red squares). The solid black lines, show the fits from the back-transformed <i>P</i>(<i>r</i>) functions <b>(b)</b> Distance distribution function, <i>P</i>(<i>r</i>), obtained from the scattering profiles shown in (a).</p

Assembly of R.EcoR124I_NT.

<p><b>(a)</b> Electrophoretic mobility shift assay (EMSA)<b>.</b> A 6% native gel was run at 150 V for 2.5 hours. Final concentrations of MTase and DNA were 2 µM. Lanes labelled R1 and R2 correspond to 1∶1 and 2∶1 molar ratios of HsdR to MTase. No difference was observed in the presence of 10 mM MgCl₂. <b>(b)</b> Dynamic light scattering. The R1 and R2 complexes had hydrodynamic radii of 6.1 and 6.2 nm, respectively. <b>(c)</b> Sedimentation coefficient distributions of R.EcoR124I_NT (the R2 complex) plus and minus DNA. Sedimentation velocity data were collected at 285 nm, scanning every 12 minutes at 10°C at 30,000 rpm. Peak sedimentation coefficients for the free enzyme (9.5 S) and the enzyme bound to DNA (10.6 S) were converted to S_20,w values of 12.6 S and 14.0 S, respectively.</p