|Budget Amount *help
¥98,600,000 (Direct Cost : ¥98,600,000)
Fiscal Year 2006 : ¥19,500,000 (Direct Cost : ¥19,500,000)
Fiscal Year 2005 : ¥19,500,000 (Direct Cost : ¥19,500,000)
Fiscal Year 2004 : ¥21,000,000 (Direct Cost : ¥21,000,000)
Fiscal Year 2003 : ¥21,000,000 (Direct Cost : ¥21,000,000)
Fiscal Year 2002 : ¥17,600,000 (Direct Cost : ¥17,600,000)
"Recoding" is non-standard genetic decoding such as translational frameshifting. Antizyme, a regulatory protein for polyamines, requires translational frameshifting for its expression and induction by polyamines. We have identified the signal sequences, namely, the shift site, a downstream pseudoknot, and an upstream sequence, for the frameshifting of AZ1, an major isoform of AZ. This project aimed to understand the mechanism of AZ1 frameshifting form the aspects of not only the cis-acting signals but also translational machinery and small-molecule regulators, and the results are summarized as the followings. (1) The molecular evolution, interchangeability with other recoding signals, and the interaction site of the polyamine action of the AZ1 frameshift signal were analyzed. A cellular binding protein for AZ1 pseudoknot has been identified. Some small nucleotides that complementarily bind to the frameshift signals are found to affect frameshift efficiency. (2) In the ribosomal hopping directed by the mammalian AZl sequence in E. coli, re-pairing of the peptidyl-tRNA is found to be major determinant of the landing codon. (3) To identify translation machinery involved in AZ1 frameshifting, genetic screening was performed in the fission yeast. We screened genomic mutations, multicopy suppressors, dominant suppressors, but could not obtain any significant candidate. (4) Some branched-chain polyamines originated from a thermophilic bacteria or acetylated forms of polyamines showed specific effect on AZ1 frameshifting. (5) To discover new recoding genes, we screed a random sequence pool in E. coli. We also searched mRNAs of which expression levels are elevated in the nonsense-mediated mRNA decay mutants in the fission yeast. Both screening brought about several candidate for new recoding. (6) Analysis of AZ1 knockout mice revealed that a phenotype of lack of frameshift regulation of AZl is embryonic death due to a disturbance of hematopoietic cell differentiation.