Gong F, Yanofsky C
Department of Biological Sciences, Stanford University, Stanford, California 94305, USA.
J Biol Chem. 2001 Jan 19;276(3):1974-83. doi: 10.1074/jbc.M008892200. Epub 2000 Oct 24.
Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Catabolite repression regulates transcription initiation, whereas excess tryptophan induces antitermination at Rho factor-dependent termination sites in the leader region of the operon. Synthesis of the leader peptide, TnaC, is essential for antitermination. BoxA and rut sites in the immediate vicinity of the tnaC stop codon are required for termination. In this paper we use an in vitro S-30 cell-free system to analyze the features of tna operon regulation. We show that transcription initiation is cyclic AMP (cAMP)-dependent and is not influenced by tryptophan. Continuation of transcription beyond the leader region requires the presence of inducing levels of tryptophan and synthesis of the TnaC leader peptide. Using a tnaA'-'trpE fusion, we demonstrate that induction results in a 15-20-fold increase in synthesis of the tryptophan-free TnaA-TrpE fusion protein. Replacing Trp codon 12 of tnaC by an Arg codon, or changing the tnaC start codon to a stop codon, eliminates induction. Addition of bicyclomycin, a specific inhibitor of Rho factor action, substantially increases basal level expression. Analyses of tna mRNA synthesis in vitro demonstrate that, in the absence of inducer transcription is terminated and the terminated transcripts are degraded. In the presence of inducer, antitermination increases the synthesis of the read-through transcript. TnaC synthesis is observed in the cell-free system. However, in the presence of tryptophan, a peptidyl-tRNA also appears, TnaC-tRNA(Pro). Our findings suggest that inducer acts by preventing cleavage of TnaC peptidyl-tRNA. The ribosome associated with this newly synthesized peptidyl-tRNA presumably stalls at the tnaC stop codon, blocking Rho's access to the BoxA and rut sites, thereby preventing termination. 1-Methyltryptophan also is an effective inducer in vitro. This tryptophan analog is not incorporated into TnaC.
大肠杆菌色氨酸酶（tna）操纵子的表达受分解代谢物阻遏和色氨酸诱导的转录抗终止调控。分解代谢物阻遏调节转录起始，而过量色氨酸在操纵子前导区的Rho因子依赖性终止位点诱导抗终止。前导肽TnaC的合成对抗终止至关重要。tnaC终止密码子紧邻的BoxA和rut位点是终止所必需的。在本文中，我们使用体外S-30无细胞系统分析tna操纵子调控的特征。我们表明转录起始是环磷酸腺苷（cAMP）依赖性的，且不受色氨酸影响。转录延伸至前导区之外需要诱导水平的色氨酸存在以及TnaC前导肽的合成。使用tnaA'-'trpE融合体，我们证明诱导导致无色氨酸的TnaA-TrpE融合蛋白合成增加15 - 20倍。将tnaC的第12位色氨酸密码子替换为精氨酸密码子，或将tnaC起始密码子变为终止密码子，可消除诱导作用。添加双环霉素，一种Rho因子作用的特异性抑制剂，可显著增加基础水平表达。体外tna mRNA合成分析表明，在没有诱导剂的情况下转录终止，且终止的转录本会被降解。在有诱导剂存在时，抗终止增加通读转录本的合成。在无细胞系统中观察到了TnaC的合成。然而，在有色氨酸存在时，也会出现一种肽基-tRNA，即TnaC-tRNA(Pro)。我们的发现表明诱导剂通过阻止TnaC肽基-tRNA的切割起作用。与这种新合成的肽基-tRNA相关的核糖体大概在tnaC终止密码子处停滞，阻止Rho因子接近BoxA和rut位点，从而防止终止。1-甲基色氨酸在体外也是一种有效的诱导剂。这种色氨酸类似物不会掺入TnaC中。
