Purification and characterization of a new factor which restores protein synthesis in  a conditionally lethal mutant of Escherichia coli.
The mutant Escherichia coli strain, N4316, has a temperature-sensitive defect in a protein factor required for translation in vitro of bacteriophage f2 RNA. We have purified the normal counterpart of this factor from a wild-type strain, using as an assay its ability to restore the activity of mutant extracts at non-permissive temperature. Our final preparation is free of known initiation, propagation, and release factors, proving that the factor is a new component required for translation. The new factor has a molecular weight of 95000 with preliminary data suggesting a subunit structure. 70% of this protein is found in the soluble-cell fraction, the rest being associated with 70-S ribosomes. Kinetic analyses indicate that the factor acts early in translation. Expression of the defect is highly dependent on the Mg2+ concentration, no temperature-sensitivity being apparent at 15 mM Mg2+. At lower Mg2+ concentrations, the defect is expressed only with natural mRNAs such as f2 RNA, and not with artifical polymers such as poly(U). This specificity suggests that the factor may function in events coded by special sequences in the natural messengers
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D002845:Chromatography; D003090:Coliphages; D003600:Cytosol / Q000032:analysis; D004926:Escherichia coli / Q000032:analysis / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005804:Genes, Lethal; D006882:Hydroxyapatites; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009154:Mutation; D012270:Ribosomes / Q000032:analysis; D013696:Temperature; D014764:Viral Proteins / Q000096:biosynthesis*
D001426:Bacterial Proteins; D006882:Hydroxyapatites; D014764:Viral Proteins; D008274:Magnesium