Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering

Light scattering and standard stopped-flow techniques were used to monitor rapid association of ribosomal subunits during initiation of eubacterial protein synthesis. The effects of the initiation factors IF1, IF2, IF3 and buffer conditions on subunit association were studied along with the role of GTP in this process. The part of light scattering theory that is essential for kinetic measurements is high-lighted in the main text and a more general treatment of Rayleigh scattering from macromolecules is given in an appendix

A New Role for Translation Initiation Factor 2 in Maintaining Genome Integrity

Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA–damaging agent is present

Evidence for turnover of polysomal structures during aminoacyl-tRNA deprivation in relaxed mutants of E. coli.

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Polysome level and stability in stringent E. coli strains under aminoacyl-tRNA deprivation.

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Differential pattern of misreading induced by streptomycin in vitro.

International audienceA simplified, plasmid-directed coupled system was used to study the effect of streptomycin on the accuracy of natural messenger translation in vitro. The interaction of six different codons with their cognate tRNAs and 18 non-cognate tRNAs was analysed in the presence and absence of the antibiotic. Streptomycin appeared to modify, to a varying extent, the frequency of errors in codon-anticodon recognition. From this observation, some rules for mistranslation were inferred.A simplified, plasmid-directed coupled system was used to study the effect of streptomycin on the accuracy of natural messenger translation in vitro. The interaction of six different codons with their cognate tRNAs and 18 non-cognate tRNAs was analysed in the presence and absence of the antibiotic. Streptomycin appeared to modify, to a varying extent, the frequency of errors in codon-anticodon recognition. From this observation, some rules for mistranslation were inferred

High fidelity of guanine translation in a plasmid-directed in vitro system.

International audienceThe extent of misreading of individual bases in the first or second codon position has been measured in vitro in a simplified plasmid-directed coupled system in which natural messenger translation is restricted to the formation of the N-terminal di- or tripeptide. Experiments were performed under conditions of competition between cognate and noncognate tRNAs in the presence of streptomycin to maximize the frequency of reading errors. A striking lack of susceptibility to mistranslation of guanine, as compared to the other 3 bases, was observed.The extent of misreading of individual bases in the first or second codon position has been measured in vitro in a simplified plasmid-directed coupled system in which natural messenger translation is restricted to the formation of the N-terminal di- or tripeptide. Experiments were performed under conditions of competition between cognate and noncognate tRNAs in the presence of streptomycin to maximize the frequency of reading errors. A striking lack of susceptibility to mistranslation of guanine, as compared to the other 3 bases, was observed

Further analysis of the polysomal casein kinase activity of rat liver.

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The stringent response to inhibition of peptide chain initiation in Escherichia coli.

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The effects of trimethoprim and erythromycin on polysome metabolism in Escherichia coli.

International audienceTrimethoprim and erythromycin were shown to have different overall effects on in vivo polysome metabolism in Escherichia coli. In a rel A+-rel A pair of strains, trimethoprim treatment induces a reduction of polysome level to a variable extent, similarly to aminoacyl tRNA deprivation of cells, but persisting polysomes remain dynamic structures in a state of continual turnover. In contrast, erythromycin stabilizes polysome level to a high value in either kind of strain, but maintained polysomes appear as "frozen" structures unable to undergo ribosome translocation.Trimethoprim and erythromycin were shown to have different overall effects on in vivo polysome metabolism in Escherichia coli. In a rel A+-rel A pair of strains, trimethoprim treatment induces a reduction of polysome level to a variable extent, similarly to aminoacyl tRNA deprivation of cells, but persisting polysomes remain dynamic structures in a state of continual turnover. In contrast, erythromycin stabilizes polysome level to a high value in either kind of strain, but maintained polysomes appear as "frozen" structures unable to undergo ribosome translocation

Protein kinase activity tightly bound to liver polysomes.

International audienceRat liver polysomes washed with 0.5-1.5 M KCl at 37 degrees C keep a constant protein kinase activity revealed only by auto-phosphorylation of ribosomal proteins. The enzyme catalyzes the transfer of the gamma-phosphate group from ATP to serine (75%) but also to threonine residues (25%). It is released when polysomes are dissociated into subunits using centrifugation through a sucrose gradient containing a high K+/Mg++ ratio. Its properties have been compared with those of the two other enzymatic activities which are, in contrast, washed out during salt treatment of polysomes. After release upon polysome dissociation, this third activity is able to phosphorylate histone II A. Protection of the enzyme in the polysome structure against salt treatment, suggests that it is located at the junction of the two subunits.Rat liver polysomes washed with 0.5-1.5 M KCl at 37 degrees C keep a constant protein kinase activity revealed only by auto-phosphorylation of ribosomal proteins. The enzyme catalyzes the transfer of the gamma-phosphate group from ATP to serine (75%) but also to threonine residues (25%). It is released when polysomes are dissociated into subunits using centrifugation through a sucrose gradient containing a high K+/Mg++ ratio. Its properties have been compared with those of the two other enzymatic activities which are, in contrast, washed out during salt treatment of polysomes. After release upon polysome dissociation, this third activity is able to phosphorylate histone II A. Protection of the enzyme in the polysome structure against salt treatment, suggests that it is located at the junction of the two subunits