Elsevier

Methods in Enzymology

Volume 30, 1974, Pages 293-303
Methods in Enzymology

[30] Mammalian release factor; in Vitro Assay and Purification

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Publisher Summary

Peptide chain termination and several of its intermediate steps can be studied in mammalian extracts. The codon directed release of peptides from ribosomal bound peptidyl-tRNA is investigated in mammalian extracts by modification of the formylmethionine (fMet) release assay described for bacterial extracts. Two additional methods for investigation of mammalian peptide chain termination are also available. The first studies fMet-tRNA hydrolysis as a partial reaction of the total process. The hydrolysis of ribosomal bound f[3H]Met-tRNA which occurs in reactions containing RF and 20% ethanol does not require terminator codon recognition and is not stimulated by GTP.

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Cited by (37)

  • Dysfunctions of the translational machinery in digestive glands of mussels exposed to mercury ions

    2013, Aquatic Toxicology
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    When complex eRF1·eRF3·GTP binds to the A-site of such a complex, [3H]Met releases from the ribosome. Therefore, such a system enables the evaluation of the efficiency of the termination process, by monitoring the reduction of radioactivity bound to the ribosome (Caskey et al., 1974). As indicated in Fig. 6A, exposure of mussels to Hg2+ causes a time-dependent reduction in the termination efficiency, which at the 15th day of the exposure reaches the 55% of the control value.

  • In Vitro Reconstitution of Eukaryotic Translation Reveals Cooperativity between Release Factors eRF1 and eRF3

    2006, Cell
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    It has been suggested that eRF3 might perform a role similar to that of prokaryotic RF3 by promoting recycling of eRF1 (Zavialov et al., 2001), but recent genetic data suggest that eRF3's GTPase activity might instead couple termination-codon recognition by eRF1 with efficient peptide release (Salas-Marco and Bedwell, 2004). The principal assay used for many years to study termination in vitro is based on artificial pre-TCs obtained by binding an AUG initiation codon and a stop signal (UAAA, UAGA, or UGAA) to “empty” ribosomes reconstituted from 40S and 60S subunits in the presence of [35S]fMet-tRNA to simulate peptidyl tRNA (Caskey et al., 1974). Release of [35S]fMet from fMet-tRNA mimics peptidyl-tRNA hydrolysis.

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This work was supported by Grant GM-18682-02.

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