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Biomedical subjects

L Bosch

Publications and source records attributed to L Bosch.

142 records · Page 8Linked to original sources
PubMed1970

Dissociation of 70 S E. coli ribosomes induced by a ribosomal factor (DF). Electrophoretic studies of the ribosomal particles.

The dissociation of purified 70 S.E. coli ribosomes, induced by the dissociation factor DF, has been studied by submitting the reaction mixtures to electrophoresis on polyacrylamide gels. The electrophoretic analysis of the ribosome mixtures revealed a heterogeneity which escaped detection by conventional sucrose gradient centrifugation. Increasing amounts of DF in the reaction mixtures converted 70 S ribosomes to particles (designated 70 S (I)) which migrate slower in the electric field than the original 70 S ribosomes. These 70 S (I) ribosomes still consist of both subunits. They dissociate upon further raising the DF concentration.

Journal Article↗
PubMed1970

An initiation factor causing dissociation of E. coli ribosomes.

Purification of crude initiation factors, essential for polypeptide synthesis in cell-free systems of E. coli, yielded a fraction DF which causes dissociation of 70 S ribosomes. Its stoichiometric action on 70 S ribosomes is antagonized by increasing Mg(2+) concentrations but not by the addition of 30 S and 50 S subunits washed with high salt concentration. GTP did not stimulate this dissociating action. 2 &mgr;g of our most purified preparation caused 100% dissociation of 100 &mgr;g of 70 S ribosomes without added GTP. DF-induced dissociation is a very rapid process at 37 degrees C and is temperature-dependent in the range of 0 degrees -37 degrees C. DF, which is thermolabile factor, is much less or not effective with complexed 70 S ribosomes bearing peptidyl-tRNA and mRNA.

Journal Article↗
PubMedDate not supplied

The influence of tRNA located at the P-site on the turnover of EF-Tu.GTP on ribosomes.

The turnover of EF-Tu.GTP on poly-U programmed ribosomes was measured both in the presence and in the absence of N-acetylated Phe-tRNA(Phe) at the P-site. The reaction was uncoupled from protein synthesis by omitting Phe-tRNA(Phe) at the A-site. In this reaction, the ribosome can be considered as an enzyme catalysing the transition of EF-Tu.GTP to EF-Tu.GTP. A constant EF-Tu.GTP concentration is maintained by regenerating GDP to GTP at the expense of phosphoenolpyruvate by pyruvate kinase. The rate constants are determined using a procedure which corrects for the reduction in specific activity of GTP due to regeneration of the nucleotide. Ribosomes with an occupied P-site are more efficient in stimulating the GTPase of EF-Tu.GTP than ribosomes with an empty P-site. The data suggest that this is mainly caused by an increased affinity of EF-Tu.GTP for ribosomes with a filled P-site rather than by an enhanced reactivity of the GTPase centre.

Binding Sites↗
PubMedDate not supplied

Antibiotic resistance mechanisms of mutant EF-Tu species in Escherichia coli.

Analysis of antibiotic-resistant EF-Tu mutants has revealed a connection between resistance and structural elements that participate in the GTPase switching mechanism. Both random and site-directed mutagenesis methods have yielded sets of purified mutant EF-Tu resistant to kirromycin (kirT) or pulvomycin (pulT). All kirT mutations cluster in the interface of domain 1 and 3 of EF-Tu in its GTP-bound conformation, not in that of EF-Tu.GDP. Other evidence also suggests that kirromycin binds to the interface of wild-type EF-Tu, thereby jamming the GTPase switch. Various functional studies reveal two subsequent resistance mechanisms. The first hinders kirromycin binding to EF-Tu.GTP and the second occurs after GTP hydrolysis by rejection of bound kirromycin. All pulT mutations cluster in the three-domain junction interface of EF-Tu. GTP (which is an open hole in EF-Tu.GDP) and destabilize a salt-bridge network. Pulvomycin may bind nearby and overlap with tRNA binding. Mutations show that a D99-R230 salt bridge is not essential for the transduction of the GTPase switch signal from domain 1. In vivo and in vitro studies reveal that pulvomycin sensitivity is dominant over resistance. This demands a revision of the current view of the mechanism of pulvomycin inhibition of protein synthesis and may support a translation model with two EF-Tus on the ribosome. Several mutant EF-Tu species display altered behaviour towards aminoacyl-tRNA with interesting effects on translational accuracy. KirT EF-Tu(A375T) is able to reverse the streptomycin-dependent phenotype of a ribosomal protein S12 mutant strain to streptomycin sensitivity.

Bacterial Proteins↗