The "eRF" clone corresponds to tryptophanyl-tRNA synthetase, not mammalian release factor.
To study the similarity between a putative cloned mammalian release factor (RF) and tryptophanyl-tRNA synthetase (TRS), a recombinant rabbit RF fusion protein was expressed from prokaryotic expression vectors. Purified fractions of the fusion proteins were tested for TRS and RF activities. Addition of the fusion protein to a TRS assay increased the binding of tryptophan to tRNA(Trp). However, in an assay for RF activity, the addition of the fusion protein resulted in release of only 1-3% of formylmethionine from an fMet-tRNA-AUG-ribosome intermediate that had been provided with UAAA as message. To confirm this result, the coding region of the putative eukaryotic RF clone "eRF" was used for in vitro transcription and translation in a rabbit reticulocyte lysate system, resulting in the synthesis of a single 56-kDa protein. The influence of this 56-kDa protein on the termination of translation directed by tobacco mosaic virus was studied. Tobacco mosaic virus RNA produced a major 126-kDa protein and a minor 184-kDa readthrough protein in an in vitro translation system. The protein generated from the "eRF" coding region did not inhibit biosynthesis of the 184-kDa readthrough virus protein. Instead, it increased the yield of both viral proteins. This increase was presumably due to its TRS activity. Chromatography of proteins derived from human lymphoblasts separated RF from TRS activity. Thus, our results indicate that the previously cloned "eRF" clone encodes TRS and that rabbit reticulocyte RF activity lies in a different protein.
Timchenko
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Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX 77030.
Caskey
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CT
D000818:Animals; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D010443:Peptide Chain Termination, Translational / Q000235:genetics / Q000502:physiology*; D014176:Protein Biosynthesis; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D014027:Tobacco Mosaic Virus / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism; D014369:Tryptophan-tRNA Ligase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D014364:Tryptophan; D014369:Tryptophan-tRNA Ligase