[Show abstract][Hide abstract] ABSTRACT: We have synthesized a series of 5'-phosphorylated and 5'-cytidylyl-(3'-5')-cytidylyl-(3'-5')-puromycin derivatives that have backbone-elongated substrates. All the synthesized puromycin derivatives showed good solubility in water and were applied to translation inhibitory assay in a reconstituted Escherichia coli translation system.
[Show abstract][Hide abstract] ABSTRACT: There have been recent advances in the ribosomal synthesis of various molecules composed of nonnatural ribosomal substrates. However, the ribosome has strict limitations on substrates with elongated backbones. Here, we show an unexpected loophole in the E. coli translation system, based on a remarkable disparity in its selectivity for beta-amino/hydroxy acids. We challenged beta-hydroxypropionic acid (beta-HPA), which is less nucleophilic than beta-amino acids but free from protonation, to produce a new repertoire of ribosome-compatible but main-chain-elongated substrates. PAGE analysis and mass-coupled S-tag assays of amber suppression experiments using yeast suppressor tRNAPheCUA confirmed the actual incorporation of beta-HPA into proteins/oligopeptides. We investigated the side-chain effects of beta-HPA and found that the side chain at position alpha and R stereochemistry of the beta-substrate is preferred and even notably enhances the efficiency of incorporation as compared to the parent substrate. These results indicate that the E. coli translation machinery can utilize main-chain-elongated substrates if the pKa of the substrate is appropriately chosen.
No preview · Article · Jun 2007 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: The ribosome catalyzes oligo/polymerization of amino acids. We designed an allowable modification of amino acid backbone, based on the hypothesized mechanism of peptidyl transfer reaction. Nonsense suppression method was used to investigate the acceptability of these substrates in the prokaryotic ribosomal system. The E.coli ribosome showed a restricted tolerance to the backbone modification, especially for main-chain elongation. However, our designed homologous beta-hydroxyalkanoic acid with elongated methylene (backbone) chain-length was revealed to be a possible substrate for the E.coli ribosome.
No preview · Article · Feb 2006 · Nucleic Acids Symposium Series
[Show abstract][Hide abstract] ABSTRACT: A molecular-beacon-type riboregulator (mRNA) was applied to multiply catalytic gene sensing. It consists of a reporter gene for firefly protein luciferase and, upstream thereof, a regulator hairpin domain composed of an RBS/anti-RBS stem (RBS = ribosome binding site) and a loop which is complementary to the target. The hairpin and, hence, the RBS are rendered open upon binding of a target oligonucleotide of the human CC chemokine receptor 5 sequence in a prokaryotic cell-free translation system (10 muL) to ignite ribosomal catalytic translation, or transcription/translation when using a DNA form of the probe, to produce luciferase, which is assayed by a catalytic chemiluminescence reaction. The sensing, using an unmodified RNA or even dsDNA as a probe with a chemiluminescence output, is thus doubly catalytic or amplifiable with a sensitivity at </=50 fmol in respect to the target with 4.5 fmol (1 ng/muL) of probe and a single nucleotide resolution.
No preview · Article · Apr 2005 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: Nonsense suppression method was used to probe the allowable modification of substrate (amino acid) backbone in the prokaryotic ribosomal system. Dihydrofolate reductase (DHFR) with an amber mutation was translated in the RF1-diminished prokaryotic cell free translation system in the presence of chemically-misacylated yeast tRNA(Phe)CUA. The prokaryotic ribosome showed a restricted tolerance to the backbone modification. Although natural-type alpha-amino acid was accepted as a good substrate for the ribosome, incorporation of beta-aminopropionic acid was not detected under our experimental conditions. Interestingly, we found that the homologous beta-hydroxyalkanoic acid with elongated methylene (backbone) chain-length can be a substrate for the ribosome, giving an important implication for the chemical mechanism of the ribosome-catalyzed peptide bond forming reaction.
No preview · Article · Feb 2005 · Nucleic Acids Symposium Series