High-Mobility-Group A-Like CarD Binds to a DNA Site Optimized for Affinity and Position and to RNA Polymerase To Regulate a Light-Inducible Promoter in Myxococcus xanthus
High-Mobility-Group A-Like CarD Binds to a DNA Site Optimized for Affinity and Position and to RNA Polymerase To Regulate a Light-Inducible Promoter in Myxococcus xanthus
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DOI：
10.1128/jb.01766-12
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发表时间：
2013-01-01
影响因子：
3.2
通讯作者：
Elias-Arnanz, Montserrat
中科院分区：
文献类型：
--
作者：
Garcia-Heras, Francisco;Abellon-Ruiz, Javier;Elias-Arnanz, Montserrat
CarD-CarG复合物控制细菌粘球菌中的各种细胞过程，包括子实体发育和光诱导的胡萝卜素生成。定义大的CarD_CdnL_TRCF蛋白家族的CarD N-末端结构域结合CarG，CarG是一种不结合DNA的锌相关蛋白。CardD的C-末端结构域类似于真核生物高迁移率族A（HMGA）蛋白，其DNA结合AT钩特异性识别适当间隔的AT丰富的道小沟。在这里，我们调查的决定因素的唯一已知的卡D结合位点，一个关键的卡D-CarG调控的启动子的carQRS操纵子（PQRS），光诱导型启动子依赖于细胞质外功能（ECF）σ因子CarQ。在体外，突变任一3 bp AT tracts的这一卡识别位点（TTTCCAGAGCTTT）损害DNA结合，转移AT tracts相对于PQRS没有影响或略有降低DNA结合，并取代天然位点的HMGA 1a结合一个在人β干扰素启动子（与较长的AT tracts）显着增强DNA结合。然而，在体内，所有这些变化阻止了野生型M的PQRS激活。xanthus，以及在具有被脱卤Anaeromyxanthus CarD-CarG（CarD（Ad）-CarG（Ad））替换的CarD-CarG对的菌株中。尽管CarD（Ad）的体外DNA结合亲和力较低，但CarD（Ad）-CarG（Ad）在功能上等同于CarD-CarG，其C-末端结构域类似于组蛋白H1而不是HMGA。我们表明，CardD物理协会与RNA聚合酶（RNAP），特别是通过与RNAP β亚基的相互作用。我们的研究结果表明，CardD调节光诱导，ECF σ依赖性启动子耦合RNAP招聘和结合到一个特定的DNA位点优化的亲和力和位置。
The CarD-CarG complex controls various cellular processes in the bacterium Myxococcus xanthus including fruiting body development and light-induced carotenogenesis. The CarD N-terminal domain, which defines the large CarD_CdnL_TRCF protein family, binds to CarG, a zinc-associated protein that does not bind DNA. The CarD C-terminal domain resembles eukaryotic high-mobility-group A (HMGA) proteins, and its DNA binding AT hooks specifically recognize the minor groove of appropriately spaced AT-rich tracts. Here, we investigate the determinants of the only known CarD binding site, the one crucial in CarD-CarG regulation of the promoter of the carQRS operon (PQRS), a light-inducible promoter dependent on the extracytoplasmic function (ECF) sigma factor CarQ. In vitro, mutating either of the 3-bp AT tracts of this CarD recognition site (TTTCCAGAGCTTT) impaired DNA binding, shifting the AT tracts relative to PQRS had no effect or marginally lowered DNA binding, and replacing the native site by the HMGA1a binding one at the human beta interferon promoter (with longer AT tracts) markedly enhanced DNA binding. In vivo, however, all of these changes deterred PQRS activation in wild-type M. xanthus, as well as in a strain with the CarD-CarG pair replaced by the Anaeromyxobacter dehalogenans CarD-CarG (CarD(Ad)-CarG(Ad)). CarD(Ad)-CarG(Ad) is functionally equivalent to CarD-CarG despite the lower DNA binding affinity in vitro of CarD(Ad), whose C-terminal domain resembles histone H1 rather than HMGA. We show that CarD physically associates with RNA polymerase (RNAP) specifically via interactions with the RNAP beta subunit. Our findings suggest that CarD regulates a light-inducible, ECF sigma-dependent promoter by coupling RNAP recruitment and binding to a specific DNA site optimized for affinity and position.