Kostelidou K, Jagura-Burdzy G, Thomas C M
School of Biological Sciences, University of Birmingham, Birmingham, Edgbaston, B15 2TT, UK.
J Mol Biol. 1998 Aug 21;281(3):453-63. doi: 10.1006/jmbi.1998.1956.
KorA protein encoded in the central control region of IncP plasmid RK2 binds to seven operators on the plasmid genome and acts as a global repressor of genes for replication and stable inheritance functions. At trfAp, the promoter for plasmid replication genes, KorA also causes derepression of trbAp, the promoter for trbA, encoding another global regulator (TrbA), which controls genes required for conjugative transfer. Both KorB, a second global repressor encoded in the central control region, and TrbA also act in the trfAp-trbAp region to down-regulate trfAp, but neither of these extra repressors allows derepression of trbAp. To initiate a functional dissection of KorA, we used random mutagenesis and a positive selection system to identify korA mutants which no longer repressed trfAp. Nine single amino acid changes were obtained, which did not affect polypeptide length or apparent stability. These clustered either in the N-terminal region of the protein (region I) or in the putative HTH motif (region II). No changes were obtained in the C-terminal region (region III). Four truncated KorA proteins, with deletions either from the N-terminal or the C-terminal end, were also screened together with the single mutants. Both the band-shift assay with trfAp DNA and the in vivo promoter-probe assays with either trfAp or trbAp showed that none of the region II mutants could bind to DNA and repress the promoter. The region I mutants with a conservative amino acid substitution retained some DNA binding and repressor activity, as well as the ability to dimerise. However, an in vivo system to detect trans-dominance of the mutants indicated that one region I point mutant together with the two N-terminally truncated mutants had lost their dimerisation ability. Deletions into the basic C terminus of KorA did not abolish dimerisation. The results implicate region I in dimerisation, region II in DNA binding and region III in a yet unspecified role, possibly interaction with other proteins such as KorB.
编码在 IncP 质粒 RK2 中央控制区域的 KorA 蛋白与质粒基因组上的七个操纵子结合，并作为复制和稳定遗传功能相关基因的全局阻遏物。在 trfAp（质粒复制基因的启动子）处，KorA 还会导致 trbAp（trbA 的启动子，trbA 编码另一种全局调节因子 TrbA，其控制接合转移所需的基因）的去阻遏。中央控制区域编码的另一种全局阻遏物 KorB 和 TrbA 也在 trfAp - trbAp 区域发挥作用，下调 trfAp，但这些额外的阻遏物都不能使 trbAp 去阻遏。为了对 KorA 进行功能剖析，我们使用随机诱变和正选择系统来鉴定不再抑制 trfAp 的 korA 突变体。获得了九个单氨基酸变化，这些变化不影响多肽长度或表观稳定性。这些变化集中在蛋白质的 N 端区域（区域 I）或假定的螺旋 - 转角 - 螺旋基序（区域 II）。C 端区域（区域 III）未出现变化。还与单突变体一起筛选了四个截短的 KorA 蛋白，它们要么从 N 端要么从 C 端缺失。用 trfAp DNA 进行的凝胶迁移实验以及用 trfAp 或 trbAp 进行的体内启动子探针实验均表明，区域 II 突变体均不能与 DNA 结合并抑制启动子。具有保守氨基酸取代的区域 I 突变体保留了一些 DNA 结合和阻遏活性以及二聚化能力。然而，一个检测突变体反式显性的体内系统表明，一个区域 I 点突变体与两个 N 端截短的突变体失去了二聚化能力。对 KorA 碱性 C 端的缺失并未消除二聚化。结果表明区域 I 参与二聚化，区域 II 参与 DNA 结合，区域 III 具有尚未明确的作用，可能与其他蛋白质如 KorB 相互作用。
