A CONSENSUS SEQUENCE FOR BINDING OF LRP TO DNA
A CONSENSUS SEQUENCE FOR BINDING OF LRP TO DNA
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DOI：
10.1128/jb.177.17.4872-4880.1995
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发表时间：
1995-09-01
影响因子：
3.2
通讯作者：
CALVO, JM
中科院分区：
文献类型：
--
作者：
CUI, YH;WANG, Q;CALVO, JM
Lrp（亮氨酸响应调节蛋白）是一种主要的调节蛋白，参与大肠杆菌中众多操纵子的表达。对于ilvIH（受Lrp正调控的操纵子之一），Lrp结合到转录起始位点上游的多个位点并激活转录。对来自两种不同生物的ilvIH DNA内12个Lrp结合位点进行比对，揭示出一个初步的共有序列AGAATT TTTATTCT（Q. Wang，M. Sacco，E. Ricca，C. T. Lago，M. DeFelice和J. M. Calvo，《分子微生物学》7：883 - 891，1993）。为了进一步表征Lrp的结合特异性，我们使用了C. Tuerk和L. Gold（《科学》249：505 - 510，1990）的SELEX方法的一种变体，从10¹²个不同的DNA分子库中鉴定出与Lrp结合的序列。我们鉴定出63个相关的与Lrp结合的DNA序列，并估计了它们对Lrp的相对结合亲和力。通过对这些序列分析得出的一个共有序列YAGHAWATTWTDCTR，其中Y = C或T，H = 非G，W = A或T，D = 非C，R = A或G，包含明显的二重对称，并且与先前定义的序列非常相似。为了验证在亮氨酸存在的情况下Lrp可能结合到不同的DNA序列子集这一想法，我们进行了第二次筛选实验，在结合反应过程中有亮氨酸存在。在有亮氨酸和无亮氨酸情况下筛选出的DNA序列是相似的，并且亮氨酸没有刺激对在有亮氨酸情况下筛选出的任何序列的结合。因此，亮氨酸不太可能改变Lrp结合的特异性。
Lrp (leucine-responsive regulatory protein) is a major regulatory protein involved in the expression of numerous operons in Escherichia coli. For ilvIH, one of the operons positively regulated by Lrp, Lrp binds to multiple sites upstream of the transcriptional start site and activates transcription. An alignment of 12 Lrp binding sites within ilvIH DNA from two different organisms revealed a tentative consensus sequence AGAAT TTTATTCT (Q. Wang, M. Sacco, E. Ricca, C. T. Lago, M. DeFelice, and J. M. Calvo, Mol. Microbiol. 7:883-891, 1993). To further characterize the binding specificity of Lrp, we used a variation of the Selex procedure of C. Tuerk and L. Gold (Science 249:505-510, 1990) to identify sequences that bound Lrp out of a pool of 10(12) different DNA molecules. We identified 63 related DNA sequences that bound Lrp and estimated their relative binding affinities for Lrp. A consensus sequence derived from analysis of these sequences, YAGHAWATTWT DCTR, where Y = C or T, H = not G, W = A or T, D = not C, and R = A or G, contains clear dyad symmetry and is very similar to the one defined earlier. To test the idea that Lrp in the presence of leucine might bind to a different subset of DNA sequences, we carried out a second selection experiment,vith leucine present during the binding reactions. DNA sequences selected in the presence or absence of leucine were similar, and leucine did not stimulate binding to any of the sequences that were selected in the presence of leucine. Therefore, it is unlikely that leucine changes the specificity of Lrp binding.