Van Delft J H, Talens A, De Jong P J, Schmidt D S, Bosch L
Department of Biochemistry, University of Leiden, The Netherlands.
Eur J Biochem. 1988 Aug 1;175(2):363-74. doi: 10.1111/j.1432-1033.1988.tb14205.x.
The mechanism underlying feedback inhibition of tufB expression has been studied in vivo by gene-dosage experiments and by gene and operon fusions involving lacZ. Raising the cellular EF-Tu content, by introducing a multicopy plasmid encoding EF-TuA into the cell, repressed the level of EF-TuB but left the content of tRNA(Thr)3, encoded by the tRNA-tufB operon, unaffected. This indicates that autoregulation of chromosomal tufB expression does not occur by modulating transcription initiation at the promoter of the tRNA-tufB operon. This conclusion is further substantiated by experiments with a tRNA':lacZ operon fusion. The molecular ratio of chromosome-borne tufA and tufB transcripts also remained unaltered under conditions of excess EF-Tu, though experiments with a tRNA-tufB':lacZ operon fusion showed a decrease of tufB transcripts. Our data further exclude drastic effects of the autogenous repressor on processing of the contranscript of the operon into monocistronic tufB RNA and on alteration of EF-TuB turnover. Two possible mechanisms remain, which cannot yet be decided between. One is modulation of EF-Tu by transcription termination either directly or indirectly by affecting antitermination. The second is translational repression. In vitro translation of transcripts derived from SP6 clones did not reveal any feedback inhibition of EF-TuB synthesis. Surprisingly, addition of EF-Tu to a coupled transcription/translation systems was found to block transcription initiation at the primary promoter of the tRNA-tufB operon by over 90%. Although this in vitro effect of EF-Tu could not be demonstrated in vivo, possibly because of a difference in higher-order structure between plasmid-borne and chromosome-borne DNA, it indicates that under certain conditions EF-Tu binds very specifically to the tRNA-tufB operon promoter or its upstream region.
通过基因剂量实验以及涉及lacZ的基因和操纵子融合，在体内研究了tufB表达反馈抑制的潜在机制。通过将编码EF-TuA的多拷贝质粒导入细胞来提高细胞内EF-Tu的含量，这会抑制EF-TuB的水平，但由tRNA-tufB操纵子编码的tRNA(Thr)3的含量不受影响。这表明染色体tufB表达的自动调节不是通过调节tRNA-tufB操纵子启动子处的转录起始来实现的。用tRNA':lacZ操纵子融合进行的实验进一步证实了这一结论。在EF-Tu过量的条件下，染色体上tufA和tufB转录本的分子比例也保持不变，尽管用tRNA-tufB':lacZ操纵子融合进行的实验显示tufB转录本减少。我们的数据进一步排除了自体阻遏物对操纵子共转录本加工成单顺反子tufB RNA以及对EF-TuB周转改变的显著影响。仍有两种可能的机制，目前尚无法在它们之间做出决定。一种是通过直接或间接影响抗终止来调节转录终止从而调控EF-Tu。另一种是翻译阻遏。对来自SP6克隆的转录本进行体外翻译未发现对EF-TuB合成有任何反馈抑制。令人惊讶的是，发现在耦合转录/翻译系统中添加EF-Tu会使tRNA-tufB操纵子初级启动子处的转录起始受阻超过90%。尽管这种EF-Tu的体外效应在体内无法得到证实，可能是因为质粒携带的DNA和染色体携带的DNA在高级结构上存在差异，但这表明在某些条件下EF-Tu非常特异性地结合到tRNA-tufB操纵子启动子或其上游区域。
