Trp repressor interaction with bromodeoxyuridine-substituted operators alters UV-induced perturbation pattern in a sequence-dependent manner.
Trp repressor interaction with bromodeoxyuridine-substituted operators alters UV-induced perturbation pattern in a sequence-dependent manner.
复制标题
Trp 阻遏蛋白与溴脱氧尿苷取代操纵子的相互作用以序列依赖性方式改变紫外线诱导的扰动模式。
DOI：
10.1021/bi00091a002
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Matthews,KS
中科院分区：
文献类型：
--
作者：
Liu,YC;Matthews,KS
1993年7月16日接收的修订版Mandarin pt ®摘要：为了探索受trp阻遏物-操纵基因相互作用影响的DNA位点，将溴脱氧尿苷（BrdU）化学掺入TrpEDCBA、TrpR和aroH操纵基因的选定胸苷位置。尽管具有高度对称的序列，但在具有TrpEDCBA、TrpR和aroH操纵子的复合物的两半中，阻遏物保护链断裂的不同模式表明了不同的局部环境。虽然在阻遏物的存在下，在操纵子的多个位点观察到保护作用，但UV照射并没有导致交联的阻遏物-操纵子复合物。这一结果表明，合适的阻遏物残基和胸苷的5-甲基之间的大沟没有紧密接触。在色氨酸阻遏物结合和紫外线照射，除了从链断裂保护，观察到在一些网站，特别是在CTAG序列中的BrdU取代的运营商的多重。这种现象（称为条带迁移）可能是由于BrdU取代的操纵基因DNA的trp阻遏物的扭曲以及随后沿着主链沿着的不同位点暴露于链断裂所致。有趣的是，两个BrdU取代的TrpEDCBA变体操作符的UV足迹显示出不同的模式时，碱基对对称性匹配的对称轴的每一侧。这些观察结果表明，蛋白质结合引起的紫外光分解模式的改变是DNA结构改变的结果，而DNA结构改变是序列依赖性的，阻遏蛋白和激活蛋白对DNA序列的特异性识别是细菌有效转录调控所必需的（Pabo & Sauer，1984）。许多特异性DNA-蛋白质相互作用已被非常详细地表征（Steitz，1990）。在此基础上，提出了直接读出和间接读出机制对特异性DNA序列识别都是重要的。在直接读出中，蛋白质氨基酸残基与大沟DNA碱基之间的氢键和非极性相互作用对DNA序列的识别至关重要。这种机制已经通过噬菌体阻遏物的X射线晶体学分析得到证实（Jordan & Pabo，1988; Aggarwal埃塔尔，1988年）。然而，在间接读出机制中，如首次提出的大肠杆菌trp阻遏物（Otwinowski et al.，1988），DNA碱基和阻遏物残基之间没有直接接触。水介导的DNA碱基和阻遏物之间的接触以及DNA糖-磷酸骨架和阻遏物之间的接触被认为介导特异性DNA识别。已经提出了关于间接读出模型所基于的结晶trp阻遏物-操纵基因复合物的特异性的问题（Brennan &马修斯，1989; Staacke等人，1990; Carey等人，1991年）。
Revised Manuscript Received July 16, 1993® abstract: In order to explore DNA sites influenced by the trp repressor-operator interaction, bromode-oxyuridine (BrdU) was chemically incorporated into the TrpEDCBA, TrpR, and aroH operators at selected thymidine positions. Different patterns of repressor protectionfrom strand scission in the two halves of complexes with the TrpEDCBA, TrpR, and aroH operators suggest different local environments despite the highly symmetric sequences. Although protection was observed at multiple sites in the operators in the presence of repressor, UV irradiation did not lead to a cross-linked repressor-operator complex. This result indicates the absence of close contactsin the major groove between suitable repressor residues and the 5-methyl of thymidines. Upon trp repressor binding and UV irradiation, in addition to protection from strand scission, multiplets were observed at some sites, notably within CTAG sequences in the BrdU-substituted operators. This phenomenon (termed band migration) may result from distortion by the trp repressor of the BrdU-substituted operator DNA and consequent exposure of different sites along the backbone to strand scission. Interestingly, UV footprinting of two BrdU-substituted TrpEDCBA variant operators showed different patterns when base pair symmetry was matched to each side of the symmetry axis. These observations suggest that alterations in the UV photolysis pattern in response to protein binding result from DNA structuralalterations that are sequence dependent.Specific recognition of DNA sequences by both repressor and activator proteins is required for effective transcriptional regulation in bacteria (Pabo & Sauer, 1984). A number of specific DNA-protein interactions have been characterized in great detail (Steitz, 1990). On the basis of these studies, it has been suggested that both direct and indirect readout mechanisms are important for specific DNA sequence rec-ognition. In direct readout, hydrogen bonds and nonpolar interactions between amino acidresidues of proteins and DNA bases in the major groove are critical in the discrimination of DNA sequences. This mechanism has been confirmed by X-ray crystallographic analysis of phage repressors (Jordan & Pabo, 1988; Aggarwal etal., 1988). However, in the indirect readout mechanism, as first proposed for Escherichia coli trp repressor (Otwinowski et al., 1988), there are no direct contacts between DNA bases and repressor residues. Both water-mediated contacts between DNA bases and repressor and contacts between the DNA sugar-phosphate backbone and repressor are considered to mediate specific DNA recognition. Questions about the specificity of the crystallized trp repressor-operator complex, on which the indirect readout model is based, have been raised (Brennan & Matthews, 1989; Staacke et al., 1990; Carey et al., 1991).