Laboratory of Molecular Biology

About: Laboratory of Molecular Biology is a facility organization based out in Cambridge, Cambridgeshire, United Kingdom. It is known for research contribution in the topics: Gene & RNA. The organization has 19395 authors who have published 24236 publications receiving 2101480 citations.

Crystal structure of an initiation factor bound to the 30S ribosomal subunit.

Abstract: Initiation of translation at the correct position on messenger RNA is essential for accurate protein synthesis. In prokaryotes, this process requires three initiation factors: IF1, IF2, and IF3. Here we report the crystal structure of a complex of IF1 and the 30S ribosomal subunit. Binding of IF1 occludes the ribosomal A site and flips out the functionally important bases A1492 and A1493 from helix 44 of 16S RNA, burying them in pockets in IF1. The binding of IF1 causes long-range changes in the conformation of H44 and leads to movement of the domains of 30S with respect to each other. The structure explains how localized changes at the ribosomal A site lead to global alterations in the conformation of the 30S subunit.

Integrative transformation of Caenorhabditis elegans

Abstract: A technique for introducing exogenous DNA into the chromosomes of the nematode Caenorhabditis elegans is presented. A cloned C. elegans amber suppressor tRNA gene, sup-7, is used as a selectable marker. The activity of this amber suppressor is selected for by injecting worms which carry an amber termination mutation in a gene (tra-3) whose function is required for fertility. Transient expression of sup-7 is evidenced by the presence of fertile (rescued) animals in the generation after injection. In a fraction of cases, these fertile animals give rise to stable suppressor lines (eight have been characterized so far). Each of the stable suppressor lines carries injected DNA sequences. The suppressor activities have been mapped to chromosomal loci, indicating that the exogenous DNA has integrated into the genome. This technique has been used to introduce a chimeric gene containing a Drosophila heat shock promoter element fused to coding sequences from the Escherichia coli beta-galactosidase gene. This chimeric gene functions and is heat inducible in the resulting stably transformed lines.

Overexpression of integral membrane proteins for structural studies

Abstract: Determination of the structure of integral membrane proteins is a challenging task that is essential to understand how fundamental biological processes (such as photosynthesis, respiration and solute translocation) function at the atomic level. Crystallisation of membrane proteins in 3D has led to the determination of four atomic resolution structures [photosynthetic reaction centres (Allen et al. 1987; Chang et al. 1991; Deisenhofer & Michel, 1989; Ermler et al. 1994); porins (Cowan et al. 1992; Schirmer et al. 1995; Weiss et al. 1991); prostaglandin H2 synthase (Picot et al. 1994); light harvesting complex (McDermott et al. 1995)], and crystals of membrane proteins formed in the plane of the lipid bilayer (2D crystals) have produced two more structures [bacteriorhodopsin (Henderson et al. 1990); light harvesting complex (Kuhlbrandt et al. 1994)].

Atomic structure of the entire mammalian mitochondrial complex I

Abstract: Mitochondrial complex I (also known as NADH:ubiquinone oxidoreductase) contributes to cellular energy production by transferring electrons from NADH to ubiquinone coupled to proton translocation across the membrane. It is the largest protein assembly of the respiratory chain with a total mass of 970 kilodaltons. Here we present a nearly complete atomic structure of ovine (Ovis aries) mitochondrial complex I at 3.9 A resolution, solved by cryo-electron microscopy with cross-linking and mass-spectrometry mapping experiments. All 14 conserved core subunits and 31 mitochondria-specific supernumerary subunits are resolved within the L-shaped molecule. The hydrophilic matrix arm comprises flavin mononucleotide and 8 iron-sulfur clusters involved in electron transfer, and the membrane arm contains 78 transmembrane helices, mostly contributed by antiporter-like subunits involved in proton translocation. Supernumerary subunits form an interlinked, stabilizing shell around the conserved core. Tightly bound lipids (including cardiolipins) further stabilize interactions between the hydrophobic subunits. Subunits with possible regulatory roles contain additional cofactors, NADPH and two phosphopantetheine molecules, which are shown to be involved in inter-subunit interactions. We observe two different conformations of the complex, which may be related to the conformationally driven coupling mechanism and to the active-deactive transition of the enzyme. Our structure provides insight into the mechanism, assembly, maturation and dysfunction of mitochondrial complex I, and allows detailed molecular analysis of disease-causing mutations.