The mechanism of tryptophan induction of tryptophanase operon expression:: Tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNAPro
The mechanism of tryptophan induction of tryptophanase operon expression:: Tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNAPro
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DOI：
10.1073/pnas.171299298
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发表时间：
2001-07-31
影响因子：
11.1
通讯作者：
Yanofsky, C
中科院分区：
文献类型：
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作者：
Gong, F;Ito, K;Yanofsky, C
大肠埃希菌色氨酸酶(TNA)操纵子的表达受分解代谢抑制和色氨酸诱导的转录反终止调控。在之前的研究中，我们使用体外S-30系统复制了在体内观察到的这个操纵子的调控特征。我们还发现，在诱导条件下，前导肽-tRNA(TNAC-肽-tRNA(Pro))没有被切割，它在S-30反应混合物中积累。在这篇文章中，我们研究了TNAC-肽-tRNA(Pro)在体外积累和切割的要求。我们发现这种肽-tRNA仍然与翻译的核糖体结合。游离色氨酸的去除和释放因子1或2的加入导致TNAC-肽-tRNA(Pro)的水解和TNAC从核糖体-mRNA复合体中释放。添加色氨酸可以防止释放因子介导的切割。与核糖体结合的TNAC-肽-tRNA(Pro)的TNAC可转移到嘌呤霉素。这种转移也被色氨酸阻断。用各种色氨酸类似物作为色氨酸替代品的试验表明，色氨酸的作用存在严格的结构要求。我们的发现表明，将色氨酸加入到含有新生的TNAC-肽-tRNA(Pro)的核糖体中，既抑制了TNAC肽-tRNA(Pro)的水解，也抑制了TNAC肽的转移。因此，相关的翻译核糖体仍然附着在前导转录本上，在那里它阻止Rho因子结合和随后的转录终止。
Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. In a previous study, we reproduced the regulatory features of this operon observed in vivo by using an in vitro S-30 system. We also found that, under inducing conditions, the leader peptidyl-tRNA (TnaC-peptidyl-tRNA(Pro)) is not cleaved; it accumulates in the S-30 reaction mixture. In this paper, we examine the requirements for TnaC-peptidyl-tRNA(Pro) accumulation and cleavage, in vitro. We show that this peptidyl-tRNA remains bound to the translating ribosome. Removal of free tryptophan and addition of release factor 1 or 2 leads to hydrolysis of TnaC-peptidyl-tRNA(Pro) and release of TnaC from the ribosome-mRNA complex. Release factor-mediated cleavage is prevented by the addition of tryptophan. TnaC of the ribosome-bound TnaC-peptidyl-tRNA(Pro) was transferable to puromycin. This transfer was also blocked by tryptophan. Tests with various tryptophan analogs as substitutes for tryptophan revealed the existence of strict structural requirements for tryptophan action. Our findings demonstrate that the addition of tryptophan to ribosomes bearing nascent TnaC-peptidyl-tRNA(Pro) inhibits both TnaC peptidyl-tRNA(Pro) hydrolysis and TnaC peptidyl transfer. The associated translating ribosome therefore remains attached to the leader transcript where it blocks Rho factor binding and subsequent transcription termination.