Structure-function analysis of three cAMP-independent forms of the cAMP receptor protein.
Structure-function analysis of three cAMP-independent forms of the cAMP receptor protein.
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cAMP 受体蛋白的三种 cAMP 独立形式的结构功能分析。
DOI：
10.1016/s0021-9258(18)66570-6
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Alan PeterkofskyS
中科院分区：
文献类型：
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作者：
James;Harman;Keith McKenneyS;Alan PeterkofskyS
cAMP受体蛋白（CRP）依赖性操纵子在大肠杆菌中的表达需要野生型CRP的CRP X cAMP复合物形式。一类crp突变体（crp*）激活不能内源性cAMP合成的菌株（cya）中的crp依赖性启动子。最重要的是crp* 突变株所表现出的调节特性的差异，其中一些突变株表现出葡萄糖介导的β-半乳糖苷酶合成抑制，而另一些突变株则没有。为了更好地了解cAMP非依赖性启动子激活和抑制的机制，我们通过克隆和DNA序列分析确定了CRP的三种CRP* 形式的一级结构;纯化了突变蛋白;表征了这些突变对CRP二级结构的影响;并在纯化的体外转录系统中研究了CRP* 激活的乳糖启动子调控。本研究的结果提供了强有力的证据表明，CRP突变改变CRP的构象，并导致cAMP非依赖性激活CRP依赖性启动子在体外。此外，CRP等位基因特异性抑制CRP* 活性的亚精胺在体外观察到平行CRP* 菌株特异性敏感性葡萄糖介导的抑制CRP依赖性酶的合成在体内。这一观察结果提供了证据表明，缺乏cAMP的细胞中的分解代谢物抑制可能是通过抑制CRP* 活性的机制介导的。
cAMP receptor protein (CRP)-dependent operon expression in Escherichia coli requires the CRP X cAMP complex form of wild-type CRP. One class of crp mutants (crp*) activates CRP-dependent promoters in strains (cya) incapable of endogenous cAMP synthesis. Of fundamental interest is the difference in regulatory properties exhibited by crp* mutant strains, some of which exhibit glucose-mediated repression of beta-galactosidase synthesis, some of which do not. To gain a better understanding of the mechanisms of cAMP-independent promoter activation and repression we have: determined through cloning and DNA sequence analysis the primary structure of three CRP* forms of CRP; purified the mutant proteins; characterized the effect of these mutations on CRP secondary structure; and studied CRP*-activated lac promoter regulation in a purified in vitro transcription system. The results of this study provide strong evidence that mutations in crp alter the conformation of CRP and result in cAMP-independent activation of CRP-dependent promoters in vitro. In addition, a CRP allele-specific inhibition of CRP* activity by spermidine was observed in vitro that parallels crp* strain-specific sensitivity to glucose-mediated repression of CRP-dependent enzyme synthesis in vivo. This observation provides evidence that catabolite repression in cells lacking cAMP may be mediated through a mechanism that inhibits CRP* activity.