Molecular Geometry of CsrA (RsmA) Binding to RNA and Its Implications for Regulated Expression
Molecular Geometry of CsrA (RsmA) Binding to RNA and Its Implications for Regulated Expression
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DOI：
10.1016/j.jmb.2009.07.034
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发表时间：
2009-09-18
影响因子：
5.6
通讯作者：
Romeo, Tony
中科院分区：
文献类型：
--
作者：
Mercante, Jeffrey;Edwards, Adrianne N.;Romeo, Tony
全局调节蛋白CsrA结合到目标转录物的5'-未翻译先导上并改变其翻译和/或稳定性。CsrA是一种对称的同型二聚体，含有两个相同的rna结合表面。用模型RNA底物进行的凝胶移位实验现在表明，CsrA可以同时结合转录物中的两个靶点（桥接或双位点结合）。虽然桥接发生在10至>= 63 nt的位点间距离，但类似于18个核苷酸（nt）的位点间距离是最佳的。近10-nt的间距降低了双位点结合的稳定性，因为第二个CsrA二聚体很容易竞争一个位点。单个CsrA蛋白的两个rna结合表面对于有效地体外抑制glgC'-'lacZ翻译融合是必不可少的，该融合在未翻译的先导蛋白中包含四个CsrA靶点。含有单个R44A替代物的异二聚体CsrA （HD-CsrA）在其突变体表面结合缺陷，但在其野生型（WT）表面正常结合RNA，其抑制效果比同二聚体WT-CsrA低14倍。此外，glgC中位于Shine-Dalgamo序列上游的一个CsrA靶点的缺失不影响HD-CsrA的调控，但降低了WT-CsrA的调控，证实了双位点结合的调控作用。最后，我们提出了一种机制，即在CsrA与其非编码RNA拮抗剂CsrB之间形成球状核糖核蛋白复合物。由于CsrB的许多靶点之间的距离比桥接的最佳位置更近，因此与非相邻位点的结合在能量上应该更有利，从而导致多个CsrA二聚体将CsrB系在观察到的球状结构中，而不是扩展的CsrA-CsrB复合物。2009爱思唯尔有限公司版权所有。
The global regulatory protein CsrA binds to the 5'-untranslated leader of target transcripts and alters their translation and/or stability. CsrA is a symmetrical homodimer containing two identical RNA-binding surfaces. Gel shift assays with model RNA substrates now show that CsrA can bind simultaneously at two target sites within a transcript (bridging or dual-site binding). An intersite distance of similar to 18 nucleotides (nt) was optimal, although bridging occurred with an intersite distance of 10 to >= 63 nt. The close 10-nt spacing reduced the stability of dual-site binding, as competition for one site by a second CsrA dimer readily occurred. Both RNA-binding surfaces of a single CsrA protein were essential for efficient in vitro repression of a glgC'-'lacZ translational fusion that contains four CsrA target sites within the untranslated leader. Heterodimeric CsrA (HD-CsrA) containing a single R44A replacement, which was defective for binding at its mutant surface but bound RNA normally at its wild-type (WT) surface, was similar to 14-fold less effective at repression than homodimeric WT-CsrA. Furthermore, deletion of a CsrA target site of glgC that lies upstream from the Shine-Dalgamo sequence did not affect regulation by HD-CsrA but decreased regulation by WT-CsrA, confirming a regulatory role of dual-site binding. Finally, we propose a mechanism whereby a globular ribonucleoprotein complex is formed between CsrA and its noncoding RNA antagonist, CsrB. Because many target sites of CsrB are located closer together than is optimal for bridging, binding to nonadjacent sites should be energetically favored, causing multiple CsrA dimers to tether CsrB into the observed globular form rather than an extended CsrA-CsrB complex. (C) 2009 Elsevier Ltd. All rights reserved.