CsrA regulates translation of the Escherichia coli carbon starvation gene, cstA, by blocking ribosome access to the cstA transcript
CsrA regulates translation of the Escherichia coli carbon starvation gene, cstA, by blocking ribosome access to the cstA transcript
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DOI：
10.1128/jb.185.15.4450-4460.2003
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发表时间：
2003-08-01
影响因子：
3.2
通讯作者：
Babitzke, P
中科院分区：
文献类型：
--
作者：
Dubey, AK;Baker, CS;Babitzke, P
CsrA是一种全局调节因子，其结合glgCAP前导转录物中的两个位点，从而阻断核糖体接近glgC Shine-Dalgarno序列。利用CsrA上游结合位点（GCACACGGAU）在大肠杆菌基因组序列中寻找可能受CsrA调控的其他基因。cstA包含与其Shine-Dalgarno序列重叠的精确匹配。cstA以前被证明是由碳饥饿诱导的，并编码肽转运蛋白。检测了野生型和csrA突变株中cstA-lacZ翻译融合体的表达。当细胞在Luria肉汤（LB）中生长时，csrA突变体中的表达水平大约高两倍，当LB补充有葡萄糖时，表达水平高5至10倍。先前显示cstA受环AMP（CAMP）-cAMP受体蛋白复合物调节并由Esigma转录（70）。我们研究了sigma（S）对cstA表达的影响，发现sigma（S）缺陷导致野生型和csrA突变株中cstA表达增加3倍;然而，CsrA依赖性调节得以保留。CsrA介导的cstA调节的机制也在体外进行了研究。交联研究表明，CsrA是一个同源二聚体。凝胶迁移率变化结果表明，CsrA特异性结合到cstA RNA，而耦合转录翻译和toeprint的研究表明，CsrA调节CstA的合成，通过抑制核糖体结合到cstA转录本。RNA足迹和边界分析揭示了三个或四个CsrA结合位点，其中一个重叠的cstA Shine-Dalgarno序列，如预测的。这些结果证实CsrA通过空间干扰核糖体结合来调节cstA的翻译。
CsrA is a global regulator that binds to two sites in the glgCAP leader transcript, thereby blocking ribosome access to the glgC Shine-Dalgarno sequence. The upstream CsrA binding site (GCACACGGAU) was used to search the Escherichia colt genomic sequence for other genes that might be regulated by CsrA. cstA contained an exact match that overlapped its Shine-Dalgarno sequence. cstA was previously shown to be induced by carbon starvation and to encode a peptide transporter. Expression of a cstA'-'lacZ translational fusion in wild-type and csrA mutant strains was examined. Expression levels in the csrA mutant were approximately twofold higher when cells were grown in Luria broth (LB) and 5- to 10-fold higher when LB was supplemented with glucose. It was previously shown that cstA is regulated by the cyclic AMP (CAMP)-cAMP receptor protein complex and transcribed by Esigma(70). We investigated the influence of sigma(S) on cstA expression and found that a sigma(S) deficiency resulted in a threefold increase in cstA expression in wild-type and csrA mutant strains; however, CsrA-dependent regulation was retained. The mechanism of CsrA-mediated cstA regulation was also examined in vitro. Cross-linking studies demonstrated that CsrA is a homodimer. Gel mobility shift results showed that CsrA binds specifically to cstA RNA, while coupled-transcription-translation and toeprint studies demonstrated that CsrA regulates CstA synthesis by inhibiting ribosome binding to cstA transcripts. RNA footprint and boundary analyses revealed three or four CsrA binding sites, one of which overlaps the cstA Shine-Dalgarno sequence, as predicted. These results establish that CsrA regulates translation of cstA by sterically interfering with ribosome binding.