| Co-Investigator(Kenkyū-buntansha) |
ISAKSSON Leif Dept.of Microbiology, Stockholm Univ., Professor, 微生物学部, 教授
BUCKINGHAM Richard Institute de Biologie Physico-Chimique, Professor, 部長
TATE Warren P. Dept.of Biochemistry, Univ.of Otago, Professor, 生化学部, 教授
KAJI Akira Dept.Microbiolgy, Univ.of Pennsylvania School of Med.Professor, 医学部, 教授
YANOFSKY Charles Dept.Biological Sciences, Stanford University, Professor, 生物科学部, 教授
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| Research Abstract |
Termination of protein synthesis takes place on the ribosomes as a response to a stop, rather than a sense, codon in the 'decoding' site (A site). Translation termination requires two classes of polypeptide release factors (RFs) : a class-I factor, codon-specific RFs (RF1 and RF2 in prokaryotes ; eRF1 in eukaryotes), and a class-II actor, non-specific RFs (RF3 in prokaryotes ; eRF3 in eukaryotes) that bind guanine nucleotides and stimulate class-I RF activity. The model of 'release factor tRNA mimicry' (Cell 87 : 147, 1996) explains how protein reads the genetic code (stop codon) by assuming a potential anticodon mimicry. We identified crucial amino acids (and motifs) of RFs that contribute to stop codon recognition. First, a mutant was isolated by mutagenizing bacterial RF2 (specific to UGA/UAA) and then selecting a suppressor of a temperature-sensitive RF1 (specific to UAG/UAA) mutation. This mutant RF2 carried a single substitution of Lys for Glu at position 167, and acquired the ab
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ility to terminate protein synthesis at UAG, in addition to UGA and UAA, showing that position 167 and its vicinity are directly involved in stop codon recognition by means of discrimination at the third-base (motif I). Second, site-directed mutagenesis of RF2 highlighted residues at positions 205, 206, 207 and 213 ; their alterations are toxic to cells and induce abnormal termination at the sense codon(s) (motif II). These dominant lethal effects are frequently suppressed by mutations in motif I, revealing the functional interaction between the two regions. Third, the specificity of RF1 and RF2 can be converted by swapping these two motifs. These results demonstrate that RE protein encodes a protein. Fourth, the tethered radical footprinting data reveal the proximity of the tip of the predicted 'anticodon-stem mimicry domain' of RE1 to the 30S decoding site, providing the topological support for the proposed molecular mimicry of RF.
Analogous to the initiation and elongation steps of translation, the termination step involves hydrolysis of GTP to GDP by RF3 or eRF3. The model of RF-tRNA mimicry predicts that class-II GTP/GDP-binding proteins, RF3 and eRF3, may be an 'EF-Tu-like' vehicle protein to bring class-I proteins to the A site of the ribosome or an 'EF-G-like' translocase protein. We obtained the data to support that eRF3 is functionally closer to EF-Tu than EF-G in eukaryotes, while RF3 is closer to EF-G than EF-Tu in prokaryotes. Less
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