Kowalczyk Magdalena, Borcz Barbara, Płochocka Danuta, Bardowski Jacek
Department of Microbial Biochemistry, Institute of Biochemistry and Biophysics PAS, Warszawa, Poland.
Acta Biochim Pol. 2007;54(1):71-8. Epub 2007 Mar 14.
During this study His-tagged CcpA protein purified under native conditions to obtain a biologically active protein was used for molecular analysis of CcpA-dependent regulation. Using electrophoretic mobility shift assays it was demonstrated that CcpA of L. lactis can bind DNA in the absence of the HPr-Ser-P corepressor and exhibits DNA-binding affinity for nucleotide sequences lacking cre sites. However, purified HPr-Ser-P protein from Bacillus subtilis was shown to slightly increase the DNA-binding capacity of the CcpA protein. It was also observed that CcpA bound to the cre box forms an apparently more stable complex than that resulting from unspecific binding. Competition gel retardation assay performed on DNA sequences from two PEP:PTS regions demonstrated that the ybhE, bglS, rheB, yebE, ptcB and yecA genes situated in these regions are most probably directly regulated by CcpA.
在本研究中，使用在天然条件下纯化以获得生物活性蛋白的His标签CcpA蛋白进行CcpA依赖性调控的分子分析。通过电泳迁移率变动分析表明，乳酸乳球菌的CcpA在没有HPr-Ser-P共阻遏物的情况下可以结合DNA，并且对缺乏cre位点的核苷酸序列表现出DNA结合亲和力。然而，来自枯草芽孢杆菌的纯化的HPr-Ser-P蛋白显示出可略微增加CcpA蛋白的DNA结合能力。还观察到，与cre框结合的CcpA形成的复合物明显比非特异性结合形成的复合物更稳定。对来自两个PEP:PTS区域的DNA序列进行的竞争凝胶阻滞分析表明，位于这些区域的ybhE、bglS、rheB、yebE、ptcB和yecA基因很可能直接受CcpA调控。
