Evidence for "pre-recruitment" as a new mechanism of transcription activation in Escherichia coli:: The large excess of SoxS binding sites per cell relative to the number of SoxS molecules per cell
Evidence for "pre-recruitment" as a new mechanism of transcription activation in Escherichia coli:: The large excess of SoxS binding sites per cell relative to the number of SoxS molecules per cell
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DOI：
10.1006/bbrc.2002.6559
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发表时间：
2002-03-08
影响因子：
3.1
通讯作者：
Wolf, RE
中科院分区：
文献类型：
--
作者：
Griffith, KL;Shah, IM;Wolf, RE
为了应对氧化还原循环化合物（如百草枯）所施加的氧化应激，大肠杆菌诱导SoxS的合成，然后激活大约100个基因的转录。SoxS依赖性转录激活的DNA结合位点，即"soxbox"，是高度简并的，表明基因组含有大量的SoxS结合位点。为了估计细胞中soxbox的数量，我们搜索了E。使用先前确定的最佳SoxS结合序列作为查询序列，在大肠杆菌基因组中寻找SoxS结合位点。在我们的搜索条件下，我们发现了12,500个与最佳结合序列相匹配的序列;这与我们先前基于信息论的估计一致，即一个大小为E.大肠杆菌基因组含有大约13，000个soxbox。因此，每个细胞有4 - 6个基因组的快速生长的细胞具有类似于65，000个soxbox。每个细胞中存在大量潜在的SoxS结合位点，这就提出了一个有趣的问题，即SoxS如何区分位于靶基因启动子内的功能性soxbox和散布在整个染色体上的大量等效但非功能性的结合位点。为了解决这个问题，我们用百草枯处理细胞，并使用Western印迹分析来确定每个细胞的SoxS积累动力学;我们还确定了SoxS激活基因表达的动力学。诱导后20 min，SoxS的丰度达到最高值2，500个分子/细胞，在接下来的1.5 h内逐渐下降至500个分子/细胞。考虑到靶基因表达的激活几乎立即开始，并且考虑到每个细胞的SoxS分子数量（2，500）与每个细胞的SoxS结合位点数量（65，000）之间的巨大差异，我们推断SoxS不太可能通过通常的“募集”途径激活转录，因为这种机制需要与soxbox数量相似的SoxS分子数量。相反，我们建议SoxS首先在溶液中与RNA聚合酶相互作用，然后二元复合物扫描染色体，以寻找包含soxbox的启动子，该启动子正确定位并定向用于转录激活。我们将这一新途径命名为"预招募"。"（C）2002年爱思唯尔科学（美国）。
In response to the oxidative stress imposed by redox-cycling compounds like paraquat, Escherichia coli induces the synthesis of SoxS, which then activates the transcription of similar to100 genes. The DNA binding site for SoxS-dependent transcription activation, the "soxbox," is highly degenerate, suggesting that the genome contains a large number of SoxS binding sites. To estimate the number of soxboxes in the cell, we searched the E. coli genome for SoxS binding sites using as query sequence the previously determined optimal SoxS binding sequence. We found similar to12,500 sequences that match the optimal binding sequence under the conditions of our search; this agrees with our previous estimate, based on information theory, that a random sequence the size of the E. coli genome contains similar to13,000 soxboxes. Thus, fast-growing cells with 4-6 genomes per cell have similar to65,000 soxboxes. This large number of potential SoxS binding sites per cell raises the interesting question of how SoxS distinguishes between the functional soxboxes located within the promoters of target genes and the plethora of equivalent but nonfunctional binding sites scattered throughout the chromosome. To address this question, we treated cells with paraquat and used Western blot analysis to determine the kinetics of SoxS accumulation per cell; we also determined the kinetics of SoxS-activated gene expression. The abundance of SoxS reached a maximum of 2,500 molecules per cell 20 min after induction and gradually declined to similar to500 molecules per cell over the next 1.5 h. Given that activation of target gene expression began almost immediately and given the large disparity between number of SoxS molecules per cell, 2,500, and the her of SoxS binding sites per cell, 65,000, we infer that SoxS is not likely to activate transcription by the usual "recruitment" pathway, as this mechanism would require a number of SoxS molecules similar to the number of soxboxes. Instead, we propose that SoxS first interacts in solution with RNA polymerase and then the binary complex scans the chromosome for promoters that contain a soxbox properly positioned and oriented for transcription activation. We name this new pathway "pre-recruitment." (C) 2002 Elsevier Science (USA).