Roles of Cyclic AMP Receptor Protein and the Carboxyl-Terminal Domain of the α Subunit in Transcription Activation of theEscherichia coli rhaBAD Operon
Roles of Cyclic AMP Receptor Protein and the Carboxyl-Terminal Domain of the α Subunit in Transcription Activation of theEscherichia coli rhaBAD Operon
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环AMP受体蛋白和α亚基羧基端结构域在大肠杆菌rhaBAD操纵子转录激活中的作用
作者：
C. Holcroft;S. Egan
大肠杆菌rhaBAD操纵子编码l-鼠李糖分解代谢酶。FullrhaBAD激活需要AraC家族激活子RhaS（结合在启动子- 35区域重叠的位点上）和环AMP受体蛋白（CRP，结合在RhaS的上游- 92.5处）。我们测试了c反应蛋白激活区（AR） 1和2中的丙氨酸替代对rhabad激活的影响。AR1和AR2中的一些（但不是全部）替换导致rhaBAD启动子融合表达的大约双重缺陷。我们还表达了整个c端结构域缺失的RNA聚合酶α亚基衍生物（α-Δ235），并检测了rhaBAD启动子融合的表达。最大的缺陷（54倍）发生在RhaS是唯一激活因子的截断启动子上，而全长启动子（RhaS + CRP）的缺陷较小（13倍）。通过对α亚基（α- ctd）羧基末端每个残基上表达丙氨酸取代的质粒文库的分析，鉴定出15个在全长启动子和截断启动子上都很重要的残基（主要位于dna结合决定子中）。只有一个全长缺失而非截断的启动子缺失，该残基位于dna结合行列式中。6个取代位点仅在RhaS单独激活的启动子处存在缺陷，这可能定义了α-CTD上的蛋白接触决定因子。总的来说，我们的结果表明，CRP与α-CTD的相互作用可能不是rhaBAD激活所必需的；然而，α-CTD确实有助于完全激活，可能通过与DNA和RhaS的相互作用。
ABSTRACT The Escherichia coli rhaBAD operon encodes the enzymes for catabolism of the sugar l-rhamnose. FullrhaBAD activation 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 onrhaBAD activation. Some, but not all, of the substitutions in both AR1 and AR2 resulted in approximately twofold defects in expression from rhaBAD promoter 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 from rhaBAD promoter 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 for rhaBAD activation; however, α-CTD does contribute to full activation, probably through interactions with DNA and possibly RhaS.
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影响因子：
5.6
作者：
Zhou,Y;Merkel,TJ;Ebright,RH
通讯作者：
Ebright,RH
影响因子：
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作者：
Wei Niu;Yuhong Zhou;Qianping Dong;Y. Ebright;R. Ebright
通讯作者：
Wei Niu;Yuhong Zhou;Qianping Dong;Y. Ebright;R. Ebright
影响因子：
5.6
作者：
EGAN, SM;SCHLEIF, RF
通讯作者：
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影响因子：
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作者：
EGAN, SM;SCHLEIF, RF
通讯作者：
SCHLEIF, RF
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DOI：
10.1073/pnas.78.6.3743
发表时间：
1981
影响因子：
11.1
作者：
Backman,K;Chen,YM;Magasanik,B
通讯作者：
Magasanik,B