Abstract The details of species- spe- cific aminoacylation in Oryza sativa mitochondrial tRNATrp by bacterial and eukaryotic (cytoplasm) tryptophanyl-tRNA synthetases (TrpRS) were inves- tigated. Seven single or multi...Abstract The details of species- spe- cific aminoacylation in Oryza sativa mitochondrial tRNATrp by bacterial and eukaryotic (cytoplasm) tryptophanyl-tRNA synthetases (TrpRS) were inves- tigated. Seven single or multiple mutations of three bases (G73, U72, A 68) were made in O. sativa mi- tochondrial tRNATrp to the corresponding nucleotides present in human tRNATrp. In vitro transcripts of these mutant genes were tryptophanylated by Bacil- lus subtilis and human tryptophanyl-tRNA syntheta- ses (TrpRS), and the kinetic parameters were deter- mined. The results showed that the aminoacylation of seven mutant transcripts by B. subtilis TrpRS was 53.33%―99.79% less efficient than that by wild-type O. sativa mitochondrial tRNATrp, but was 4―330 times more efficient than that by human TrpRS. The mutant MPH7 (G73, U72 and C68 in O. sativa mito- chondrial tRNA were all replaced by the counterpart residues from human tRNATrp and showed a great change in aminoacylation efficiency. Our results in- dicate that the species-specific identity elements of O. sativa mitochondrial tRNATrp are similar to bacterial and eukaryotic (cytoplasm). They are mainly located at the discriminator base, the first and the fifth pairs of bases, the discriminator base G73, two bases in the acceptor stem G1/U72 and U5/A68. Our results also provide new data in support of the hypothesis that mitochondrial tRNATrp is of eubacterial origin.展开更多
Transforming growth factorβ1 (TGFβ1) is known to be intimately involved inmany cellular processes. To explore the mechanism of TGFβ1 in these processes, the non-chimerichammerhead ribozyme and U1 snRNA chimeric rib...Transforming growth factorβ1 (TGFβ1) is known to be intimately involved inmany cellular processes. To explore the mechanism of TGFβ1 in these processes, the non-chimerichammerhead ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 were designed to down-regulateTGFβ1 expression. The activity of non-chimeric ribozyme and U1 snRNA chimeric ribozyme againstTGFβ1 in vitro and in activated hepatic stellate cells (HSCs) was detected. Cleavage reactions ofboth ribozymes in vitro demonstrated that non-chimeric ribozyme possessed better cleavage activityin vitro than U1 snRNA chimeric ribozyme. The further study showed U1 snRNA chimeric ribozymeinhibited TGFβ1 expression more efficiently than non-chimeric ribozyme in transfected HSC cells. Soit indicates that the U1 snRNA chimeric ribozyme provides an alternative approach for the researchon the precise mechanism of TGFβ1 in many cellular processes and a potential therapeutic candidatefor TGFβ1-related diseases.展开更多
基金supported by the Natural Science Foundation of Zhejiang Province(Grant No.302103).
文摘Abstract The details of species- spe- cific aminoacylation in Oryza sativa mitochondrial tRNATrp by bacterial and eukaryotic (cytoplasm) tryptophanyl-tRNA synthetases (TrpRS) were inves- tigated. Seven single or multiple mutations of three bases (G73, U72, A 68) were made in O. sativa mi- tochondrial tRNATrp to the corresponding nucleotides present in human tRNATrp. In vitro transcripts of these mutant genes were tryptophanylated by Bacil- lus subtilis and human tryptophanyl-tRNA syntheta- ses (TrpRS), and the kinetic parameters were deter- mined. The results showed that the aminoacylation of seven mutant transcripts by B. subtilis TrpRS was 53.33%―99.79% less efficient than that by wild-type O. sativa mitochondrial tRNATrp, but was 4―330 times more efficient than that by human TrpRS. The mutant MPH7 (G73, U72 and C68 in O. sativa mito- chondrial tRNA were all replaced by the counterpart residues from human tRNATrp and showed a great change in aminoacylation efficiency. Our results in- dicate that the species-specific identity elements of O. sativa mitochondrial tRNATrp are similar to bacterial and eukaryotic (cytoplasm). They are mainly located at the discriminator base, the first and the fifth pairs of bases, the discriminator base G73, two bases in the acceptor stem G1/U72 and U5/A68. Our results also provide new data in support of the hypothesis that mitochondrial tRNATrp is of eubacterial origin.
文摘Transforming growth factorβ1 (TGFβ1) is known to be intimately involved inmany cellular processes. To explore the mechanism of TGFβ1 in these processes, the non-chimerichammerhead ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 were designed to down-regulateTGFβ1 expression. The activity of non-chimeric ribozyme and U1 snRNA chimeric ribozyme againstTGFβ1 in vitro and in activated hepatic stellate cells (HSCs) was detected. Cleavage reactions ofboth ribozymes in vitro demonstrated that non-chimeric ribozyme possessed better cleavage activityin vitro than U1 snRNA chimeric ribozyme. The further study showed U1 snRNA chimeric ribozymeinhibited TGFβ1 expression more efficiently than non-chimeric ribozyme in transfected HSC cells. Soit indicates that the U1 snRNA chimeric ribozyme provides an alternative approach for the researchon the precise mechanism of TGFβ1 in many cellular processes and a potential therapeutic candidatefor TGFβ1-related diseases.
