A simplified reconstitution of mRNA-directed peptide synthesis: Activity of the epsilon enhancer and an unnatural amino acid

被引:46
作者
Forster, AC
Weissbach, H
Blacklow, SC
机构
[1] Brigham & Womens Hosp, Dept Pathol, Boston, MA 02115 USA
[2] Harvard Univ, Sch Med, Boston, MA 02115 USA
[3] Florida Atlantic Univ, Ctr Mol Biol & Biotechnol, Boca Raton, FL 33431 USA
关键词
translation; reconstitution; E; coli; ribosome; initiation factor; elongation factor; tRNA; enhancer; epsilon; peptide; unnatural amino acid; biotin;
D O I
10.1006/abio.2001.5329
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 2001 Academic Press.
引用
收藏
页码:60 / 70
页数:11
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