How mutations in tRNA distant from the anticodon affect the fidelity of decoding

The ribosome converts genetic information into protein by selecting aminoacyl tRNAs whose anticodons base-pair to an mRNA codon. Mutations in the tRNA body can perturb this process and affect fidelity. The Hirsh suppressor is a well-studied tRNA^(Trp) harboring a G24A mutation that allows readthrough of UGA stop codons. Here we present crystal structures of the 70S ribosome complexed with EF-Tu and aminoacyl tRNA (native tRNA^(Trp), G24A tRNA^(Trp) or the miscoding A9C tRNA^(Trp)) bound to cognate UGG or near-cognate UGA codons, determined at 3.2-Å resolution. The A9C and G24A mutations lead to miscoding by facilitating the distortion of tRNA required for decoding. A9C accomplishes this by increasing tRNA flexibility, whereas G24A allows the formation of an additional hydrogen bond that stabilizes the distortion. Our results also suggest that each native tRNA will adopt a unique conformation when delivered to the ribosome that allows accurate decoding

The isolation of novel "Erwinia" phages and their use in the study of bacterial phytopathogenicity

A number of bacteriophages were isolated on the "soft rot" phytopathogens Erwinia carotovora subsp. atroseptica SCRI1043 and Erwinia carotovora subsp. carotovora SCRI193. Several of these phages were used to obtain phage resistant mutants of SCRI1043, in order to investigate the role of the bacterial cell surface in virulence. While a number of phenotypic properties relating to pathogenicity and virulence of this strain have already been uncovered, little is known about the role of the cell surface in virulence. It was hoped that the use of phages would allow selection of mutants altered in both cell surface and virulence. Two phage resistant mutants, A5/22 and A5/8, exhibited reduced virulence when inoculated into potato plants, and were investigated further. Both mutants showed pleiotropic phenotypes. As well as reduced virulence and phage resistance, these mutants showed a number of other phenotypic alterations including, a reduction in the production of plant cell wall degrading enzymes, increased sensitivity to surface active agents, alterations in lipopolysaccharide and outer membrane protein profiles and reduced motility. A5/22 also exhibited bacteriostasis in the presence of galactose. Mutant A5/22 was more severely affected in its virulence than A5/8, which reflected in its greater deviation from the wild type phenotype. While no one phenotypic alteration could be directly associated with the reduced virulence of either mutant, a combination of several phenotypes may have been responsible. The phages isolated in this study were the first reported for these strains of Erwinia, and were therefore characterised under a number of criteria. All phages were grouped on the basis of structural morphology, restriction endonuclease digestion and host range. This is the first detailed characterisation of phages for Erwinia carotovora subsp. atroseptica. All isolated phages were tested for generalised transduction, a method of molecular genetic analysis so far unavailable to Erwinia carotovora subsp. atroseptica SCRI1043 and Erwinia carotovora subsp. carotovora SCRI193. Two phages, ØKP and ØMl, were capable of generalised transduction in SCRI193 and SCRI1043 respectively. Both these phages were characterised and transducing frequencies improved. ØMl is the first transducing phage reported for Erwinia carotovora subsp. atroseptica and ØKP is only the second for Erwinia carotovora subsp. carotovora. Both phages are now being used extensively in the laboratory

Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics

Cell-Sized confinement in microspheres accelerates the reaction of gene expression

Cell-sized water-in-oil droplet covered by a lipid layer was used to understand how lipid membranes affect biochemical systems in living cells. Here, we report a remarkable acceleration of gene expression in a cell-sized water-in-oil droplet entrapping a cell-free translation system to synthesize GFP (green fluorescent protein). The production rate of GFP (VGFP) in each droplet remained almost constant at least for on the order of a day, which implies 0th-order reaction kinetics. Interestingly, VGFP was inversely proportional to radius of droplets (R) when R is under 50 μm, and VGFP in droplets with R ∼ 10 μm was more than 10 times higher than that in the bulk. The acceleration rates of GFP production in cell-sized droplets strongly depended on the lipid types. These results demonstrate that the membrane surface has the significant effect to facilitate protein production, especially when the scale of confinement is on the order of cell-size

Beta-decay in odd-A and even-even proton-rich Kr isotopes

Beta-decay properties of proton-rich odd-A and even-even Krypton isotopes are studied in the framework of a deformed selfconsistent Hartree-Fock calculation with density-dependent Skyrme forces, including pairing correlations between like nucleons in BCS approximation. Residual spin-isospin interactions are consistently included in the particle-hole and particle-particle channels and treated in Quasiparticle Random Phase Approximation. The similarities and differences in the treatment of even-even and odd-A nuclei are stressed. Comparison to available experimental information is done for Gamow-Teller strength distributions, summed strengths, and half-lives. The dependence of these observables on deformation is particularly emphasized in a search for signatures of the shape of the parent nucleus.Comment: 29 pages, 16 figure

Channeling by Proximity: The Catalytic Advantages of Active Site Colocalization Using Brownian Dynamics

Nature often colocalizes successive steps in a metabolic pathway. Such organization is predicted to increase the effective concentration of pathway intermediates near their recipient active sites and to enhance catalytic efficiency. Here, the pathway of a two-step reaction is modeled using a simple spherical approximation for the enzymes and substrate particles. Brownian dynamics are used to simulate the trajectory of a substrate particle as it diffuses between the active site zones of two different enzyme spheres. The results approximate distances for the most effective reaction pathways, indicating that the most effective reaction pathway is one in which the active sites are closely aligned. However, when the active sites are too close, the ability of the substrate to react with the first enzyme was hindered, suggesting that even the most efficient orientations can be improved for a system that is allowed to rotate or change orientation to optimize the likelihood of reaction at both sites

The Crystal Structure of the Ribosome Bound to EF-Tu and Aminoacyl-tRNA

The ribosome selects a correct transfer RNA (tRNA) for each amino acid added to the polypeptide chain, as directed by messenger RNA. Aminoacyl-tRNA is delivered to the ribosome by elongation factor Tu (EF-Tu), which hydrolyzes guanosine triphosphate (GTP) and releases tRNA in response to codon recognition. The signaling pathway that leads to GTP hydrolysis upon codon recognition is critical to accurate decoding. Here we present the crystal structure of the ribosome complexed with EF-Tu and aminoacyl-tRNA, refined to 3.6 angstrom resolution. The structure reveals details of the tRNA distortion that allows aminoacyl-tRNA to interact simultaneously with the decoding center of the 30S subunit and EF-Tu at the factor binding site. A series of conformational changes in EF-Tu and aminoacyl-tRNA suggests a communication pathway between the decoding center and the guanosine triphosphatase center of EF-Tu

The Crystal Structure of the Ribosome Bound to EF-Tu and Aminoacyl-tRNA

The ribosome selects a correct transfer RNA (tRNA) for each amino acid added to the polypeptide chain, as directed by messenger RNA. Aminoacyl-tRNA is delivered to the ribosome by elongation factor Tu (EF-Tu), which hydrolyzes guanosine triphosphate (GTP) and releases tRNA in response to codon recognition. The signaling pathway that leads to GTP hydrolysis upon codon recognition is critical to accurate decoding. Here we present the crystal structure of the ribosome complexed with EF-Tu and aminoacyl-tRNA, refined to 3.6 angstrom resolution. The structure reveals details of the tRNA distortion that allows aminoacyl-tRNA to interact simultaneously with the decoding center of the 30S subunit and EF-Tu at the factor binding site. A series of conformational changes in EF-Tu and aminoacyl-tRNA suggests a communication pathway between the decoding center and the guanosine triphosphatase center of EF-Tu

SecM-Stalled Ribosomes Adopt an Altered Geometry at the Peptidyl Transferase Center

A structure of a ribosome stalled during translation of the SecM peptide provides insight into the mechanism by which the large subunit active site is inactivated

A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome

Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes