| Budget Amount *help |
¥22,300,000 (Direct Cost: ¥22,300,000)
Fiscal Year 2000: ¥10,600,000 (Direct Cost: ¥10,600,000)
Fiscal Year 1999: ¥11,700,000 (Direct Cost: ¥11,700,000)
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| Research Abstract |
The 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). Polypeptide release factors (RFs) play an essential role in this process. Although the termination process and the RF activity were discovered in vitro in the late 1960's, much of the mechanism and the apparatus have remained obscure. The pioneer works by Caskey, Capecchi, Nirenberg and colleagues have uncovered the involvement of codon-specific RFs, RF1 (for UAG/UAA) and RF2 (for UGA/UAA), of Escherichia coli in vitro. However, evidence has been lacking for direct contact between RFs and stop codons until recently. During the course of this study, we have discovered a peptide determinant in RFs equivalent to the anticodon of tRNA.Swaps of each of the conserved domains between RF1 and RF2 led to the identification of a domain that could switch recognition specificity. Systematic genetic and biochemical analyses showed that the tripeptide
… More
s Pro-Ala-Thr in RF1 and Ser-Pro-Phe in RF2 determine RF specificity. The first and third amino acids independently discriminate the second and third purine bases, respectively, hence referred to as a tripeptide 'anticodon'. It is surprising that it took 4 decades since the discovery of the genetic code to figure out the basic mechanisms behind the deciphering of its 64 codons.
After release of nascent polypeptides, the posttermination complex composed of the ribosome, deacylated tRNA, RF and mRNA needs to be dissociated for the next round of protein synthesis. Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the posttermination complex. The crystal structure of RRF has recently been solved by three groups including my group. These three molecules are composed of two domains, domain 1 and domain 2, bridged by two loops (a hinge), and superimpose almost perfectly with tRNA^<Phe> except for the amino acid-binding 3' end. It has been proposed that RRF is a near perfect tRNA mimic to explain the mechanistic disassembly of the posttermination ribosomal complex. RRF, however, is architecturally different from tRNA in that the hinge of RRF forms a flexible 'gooseneck' elbow, while the elbow of tRNA is rigid, and that this flexibility of RRF is vital for its function. We assume that nature may not have created such protein of a tRNA mimic to simply substitute for tRNA unless protein is required to pursue some function(s) that tRNA cannot do. Less
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