title: Unexpected preference of the E. coli translation system for the ester bond during incorporation of backbone-elongated substrates.
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.
authors: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan. ssando@sbchem.kyoto-u.ac.jp
authors: Shinsuke
authors: S
authors: Sando
authors: Kenji
authors: K
authors: Abe
authors: Nobuhiko
authors: N
authors: Sato
authors: Toshihiro
authors: T
authors: Shibata
authors: Keigo
authors: K
authors: Mizusawa
authors: Yasuhiro
authors: Y
authors: Aoyama
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D004952:Esters; D019344:Lactic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D009842:Oligopeptides / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D004952:Esters; D009842:Oligopeptides; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D019344:Lactic Acid; C031601:hydracrylic acid