Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700, USA.
J Mol Biol. 2013 Oct 9;425(19):3662-77. doi: 10.1016/j.jmb.2012.12.010. Epub 2012 Dec 28.
Riboswitches are cis-acting mRNA elements that regulate gene expression in response to ligand binding. Recently, a class of riboswitches was proposed to respond to the molybdenum cofactor (Moco), which serves as a redox center for metabolic enzymes. The 5' leader of the Escherichia coli moaABCDE transcript exemplifies this candidate riboswitch class. This mRNA encodes enzymes for Moco biosynthesis, and moaA expression is feedback inhibited by Moco. Previous RNA-seq analyses showed that moaA mRNA copurified with the RNA binding protein CsrA (carbon storage regulator), suggesting that CsrA binds to this RNA in vivo. Among its global regulatory roles, CsrA represses stationary phase metabolism and activates central carbon metabolism. Here, we used gel mobility shift analysis to determine that CsrA binds specifically and with high affinity to the moaA 5' mRNA leader. Northern blotting and studies with a series of chromosomal lacZ reporter fusions showed that CsrA posttranscriptionally activates moaA expression without altering moaA mRNA levels, indicative of translation control. Deletion analyses, nucleotide replacement studies and footprinting with CsrA-FeBABE identified two sites for CsrA binding. Toeprinting assays suggested that CsrA binding causes changes in moaA RNA structure. We propose that the moaA mRNA leader forms an aptamer, which serves as a target of posttranscriptional regulation by at least two different factors, Moco and the protein CsrA. While we are not aware of similar dual posttranscriptional regulatory mechanisms, additional examples are likely to emerge.
Riboswitches 是一种顺式作用的 mRNA 元件，可响应配体结合调节基因表达。最近，有人提出一类 riboswitches 可以响应钼辅因子（Moco），Moco 是代谢酶的氧化还原中心。大肠杆菌 moaABCDE 转录物的 5' 前导区是这种候选 riboswitch 类别的范例。该 mRNA 编码 Moco 生物合成的酶，而 moaA 的表达受 Moco 的反馈抑制。先前的 RNA-seq 分析表明，moaA mRNA 与 RNA 结合蛋白 CsrA（碳储存调节剂）共纯化，表明 CsrA 在体内与该 RNA 结合。在其全局调控作用中，CsrA 抑制静止期代谢并激活中心碳代谢。在这里，我们使用凝胶迁移分析来确定 CsrA 特异性地与高亲和力结合到 moaA 5' mRNA 前导区。Northern 印迹和一系列染色体 lacZ 报告基因融合研究表明，CsrA 在后转录水平上激活 moaA 表达，而不改变 moaA mRNA 水平，表明翻译控制。缺失分析、核苷酸替换研究和 CsrA-FeBABE 足迹分析确定了 CsrA 结合的两个位点。Toeprinting 测定表明 CsrA 结合导致 moaA RNA 结构发生变化。我们提出 moaA mRNA 前导区形成适体，作为至少两种不同因素（Moco 和蛋白质 CsrA）的转录后调控的靶标。虽然我们不知道类似的双重转录后调控机制，但可能会出现更多的例子。
