Roles of cyclic AMP receptor protein and the carboxyl-terminal domain of the alpha subunit in transcription activation of the Escherichia coli rhaBAD operon.
Roles of cyclic AMP receptor protein and the carboxyl-terminal domain of the alpha subunit in transcription activation of the Escherichia coli rhaBAD operon.
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环 AMP 受体蛋白和 α 亚基的羧基末端结构域在大肠杆菌 rhaBAD 操纵子转录激活中的作用。
DOI：
10.1128/jb.182.12.3529-3535.2000
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发表时间：
2000
影响因子：
3.2
通讯作者：
Egan,SM
中科院分区：
文献类型：
--
作者：
Holcroft,CC;Egan,SM
大肠杆菌rhaBAD操纵子编码用于糖-鼠李糖分解代谢的酶。 FullrhaBAD 激活需要 AraC 家族激活剂 RhaS（与启动子 -35 区域重叠的位点结合）和环 AMP 受体蛋白（CRP；在 -92.5 处紧邻 RhaS 上游结合）。我们测试了 CRP 激活区 (AR) 1 和 2 中的丙氨酸取代对 rhaBAD 激活的影响。 AR1 和 AR2 中的一些（但不是全部）替换导致 rhaBAD 启动子融合的表达出现大约两倍的缺陷。我们还表达了删除了整个 C 末端结构域的 RNA 聚合酶 α 亚基的衍生物 (α-Δ235)，并分析了 rhaBAD 启动子融合的表达。最大的缺陷（54 倍）发生在截短的启动子处，其中 RhaS 是唯一的激活剂，而全长启动子（RhaS 加 CRP）处的缺陷较小（13 倍）。对在 α 亚基 (α-CTD) 羧基末端结构域的每个残基上表达丙氨酸取代的质粒文库进行分析，鉴定出 15 个残基（主要在 DNA 结合决定簇中），这些残基对于全长和截短的启动子都很重要。全长只有一个取代有缺陷，但截短的启动子没有缺陷，该残基位于 DNA 结合决定簇中。六个取代仅在仅由 RhaS 激活的启动子处有缺陷，这些可能定义了 α-CTD 上的蛋白质接触决定簇。总体而言，我们的结果表明，rhaBAD 激活可能不需要 CRP 与 α-CTD 的相互作用；然而，α-CTD 确实有助于完全激活，可能是通过与 DNA 和可能的 RhaS 相互作用。
TheEscherichia coli rhaBADoperon encodes the enzymes for catabolism of the sugarl-rhamnose. FullrhaBADactivation requires the AraC family activator RhaS (bound to a site that overlaps the −35 region of the promoter) and the cyclic AMP receptor protein (CRP; bound immediately upstream of RhaS at −92.5). We tested alanine substitutions in activating regions (AR) 1 and 2 of CRP for their effect onrhaBADactivation. Some, but not all, of the substitutions in both AR1 and AR2 resulted in approximately twofold defects in expression fromrhaBADpromoter fusions. We also expressed a derivative of the α subunit of RNA polymerase deleted for the entire C-terminal domain (α-Δ235) and assayed expression fromrhaBADpromoter fusions. The greatest defect (54-fold) occurred at a truncated promoter where RhaS was the only activator, while the defect at the full-length promoter (RhaS plus CRP) was smaller (13-fold). Analysis of a plasmid library expressing alanine substitutions at every residue in the carboxyl-terminal domain of the α subunit (α-CTD) identified 15 residues (mostly in the DNA-binding determinant) that were important at both the full-length and truncated promoters. Only one substitution was defective at the full-length but not the truncated promoter, and this residue was located in the DNA-binding determinant. Six substitutions were defective only at the promoter activated by RhaS alone, and these may define a protein-contacting determinant on α-CTD. Overall, our results suggest that CRP interaction with α-CTD may not be required forrhaBADactivation; however, α-CTD does contribute to full activation, probably through interactions with DNA and possibly RhaS.