title: A simplified reconstitution of mRNA-directed peptide synthesis: activity of the epsilon enhancer and an unnatural amino acid.
abstract: The study of the early events in translation would be greatly facilitated by reconstitution with easily purified components. Here, Escherichia coli oligopeptide synthesis has been reconstituted using five purified recombinant His-tagged E. coli initiation and elongation factors. Highly purified ribosomes are required to yield products with strong dependencies on the translation factors. Based on HPLC separation of radiolabeled translation products from an mRNA encoding a tetrapeptide, approximately 80% of peptide products are full length, and the remaining 20% are the dipeptide and tripeptide products resulting from pausing or premature termination. Oligopeptide synthesis is enhanced when a commonly used epsilon (enhancer of protein synthesis initiation) sequence is included in the mRNA. The system incorporates a selectable, large, unnatural amino acid and may ultimately form the basis of a pure translation display technology for the directed evolution of peptidomimetic ligands and drug candidates. The recombinant clones can be exploited to prepare initiation factors and initiation complexes for structural studies, to study initiation and elongation in ribosomal peptide synthesis, and to screen for eubacterial-specific drugs.
authors.affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, 75 Francis Street, Boston, Massachusetts 02115, USA. aforster@rics.bwh.harvard.edu
authors.forename: A C
authors.initials: AC
authors.lastname: Forster
authors.forename: H
authors.initials: H
authors.lastname: Weissbach
authors.forename: S C
authors.initials: SC
authors.lastname: Blacklow
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D017123:Bacteriophage T7 / Q000235:genetics*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010452:Peptide Biosynthesis; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D010445:Peptide Elongation Factors / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism; D013912:Threonine / Q000031:analogs & derivatives / Q000378:metabolism; D013997:Time Factors; D014633:Valine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D000596:Amino Acids; D010445:Peptide Elongation Factors; D010448:Peptide Initiation Factors; D010455:Peptides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013912:Threonine; D006639:Histidine; D014633:Valine; D008239:Lysine