Open AccessPurification and Characterization of a New Factor which Restores Protein Synthesis in a Conditionally Lethal Mutant of Escherichia coli
Author(s) -
MEER John P.,
GANOZA M. Clelia
Publication year - 1975
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1975.tb04132.x
Subject(s) - ribosome , translation (biology) , mutant , escherichia coli , protein biosynthesis , biology , initiation factor , bacteriophage ms2 , messenger rna , rna , microbiology and biotechnology , eukaryotic translation , in vitro , protein subunit , bacteriophage , biochemistry , gene
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 dependant on the Mg 2+ concentration, no temperature‐sensitivity being apparent at 15 mM Mg 2+ . At lower Mg 2+ 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.