Das A, Urbanowski J, Weissbach H, Nestor J, Yanofsky C
Proc Natl Acad Sci U S A. 1983 May;80(10):2879-83. doi: 10.1073/pnas.80.10.2879.
We used an in vitro DNA-dependent protein-synthesizing system to demonstrate de novo synthesis of the leader peptide specified by the tryptophan (trp) operons of several bacterial species. Peptide synthesis was directed by self-ligated short restriction fragments containing the trp promoter and leader regions. Synthesis of leader peptides was established by demonstrating that they were labeled in vitro only by those amino acids predicted to be present in the peptides. Leader peptide synthesis was abolished by the addition of the Escherichia coli trp repressor. The E. coli trp leader peptide was found to be extremely labile in vitro; it had a half-life of 3-4 min. In a highly purified DNA-dependent peptide-synthesizing system, synthesis of the di- and tripeptides predicted from the Salmonella typhimurium trp operon leader sequence, fMet-Ala and fMet-Ala-Ala, also was observed. Using this dipeptide synthesis system, we demonstrated that translation initiation at the ribosome binding site used for trp leader peptide synthesis was reduced 10-fold when the transcript contained a segment complementary to the ribosome binding site.
我们使用体外依赖DNA的蛋白质合成系统来证明几种细菌物种的色氨酸（trp）操纵子所指定的前导肽的从头合成。肽合成由包含trp启动子和前导区域的自连接短限制片段指导。通过证明前导肽仅在体外被预测存在于肽中的那些氨基酸标记来确定前导肽的合成。添加大肠杆菌trp阻遏物可消除前导肽合成。发现大肠杆菌trp前导肽在体外极其不稳定；其半衰期为3至4分钟。在高度纯化的依赖DNA的肽合成系统中，还观察到了由鼠伤寒沙门氏菌trp操纵子前导序列预测的二肽和三肽fMet-Ala和fMet-Ala-Ala的合成。使用该二肽合成系统，我们证明当转录本包含与核糖体结合位点互补的片段时，用于trp前导肽合成的核糖体结合位点处的翻译起始减少了10倍。
