CONSTITUTIVELY SIGNALING FRAGMENTS OF TSR, THE ESCHERICHIA-COLI SERINE CHEMORECEPTOR
CONSTITUTIVELY SIGNALING FRAGMENTS OF TSR, THE ESCHERICHIA-COLI SERINE CHEMORECEPTOR
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DOI：
10.1128/jb.176.20.6340-6348.1994
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发表时间：
1994-10-01
影响因子：
3.2
通讯作者：
PARKINSON, JS
中科院分区：
文献类型：
--
作者：
AMES, P;PARKINSON, JS
Tsr是大肠杆菌的丝氨酸化学受体，具有两种信号传导模式。一个增强顺时针（CW）鞭毛旋转，和其他增强逆时针（CCW）旋转。为了确定负责这些活动的Tsr分子的部分，我们分离出Tsr胞质结构域的可溶性片段，其可以改变未受刺激的野生型细胞的鞭毛旋转模式。来自野生型Tsr的残基290至470产生CW信号，而具有单个氨基酸置换（丙氨酸313至缬氨酸）的相同片段产生CCW信号。通过上位性分析鉴定了表达这些Tsr片段信号所需的趋化性磷酸化系统的可溶性组分。与全长受体一样，这些片段似乎通过与CheA自激酶和CheW偶联因子的相互作用产生信号。CheA是所需的两个信号活动，而CheW只需要CW信号。还检查了纯化的Tsr片段在体外对CheA自磷酸化活性的影响。与体内研究结果一致，CW片段刺激CheA，而CCW片段抑制CheA。咀嚼需要刺激，但不是抑制。这些发现表明Tsr胞质结构域的180个残基的区段可以产生两个活性信号。CCW信号涉及受体和CheA激酶之间的直接接触，而CW信号也需要CheW的参与。Tsr信号片段对CheA活性的体外效应与其体内行为效应之间的相关性为趋化信号的磷酸化模型提供了令人信服的支持。
Tsr, the serine chemoreceptor of Escherichia coli, has two signaling modes. One augments clockwise (CW) flagellar rotation, and the other augments counterclockwise (CCW) rotation. To identify the portion of the Tsr molecule responsible for these activities, we isolated soluble fragments of the Tsr cytoplasmic domain that could alter the flagellar rotation patterns of unstimulated wild-type cells. Residues 290 to 470 from wild-type Tsr generated a CW signal, whereas the same fragment with a single amino acid replacement (alanine 313 to valine) produced a CCW signal. The soluble components of the chemotaxis phosphorelay system needed for expression of these Tsr fragment signals were identified by epistasis analysis. Like full-length receptors, the fragments appeared to generate signals through interactions with the CheA autokinase and the CheW coupling factor. CheA was required for both signaling activities, whereas CheW was needed only for CW signaling. Purified Tsr fragments were also examined for effects on CheA autophosphorylation activity in vitro. Consistent with the in vivo findings, the CW fragment stimulated CheA, whereas the CCW fragment inhibited CheA. CheW was required for stimulation but not for inhibition. These findings demonstrate that a 180-residue segment of the Tsr cytoplasmic domain can produce two active signals. The CCW signal involves a direct contact between the receptor and the CheA kinase, whereas the CW signal requires participation of CheW as well. The correlation between the in vitro effects of Tsr signaling fragments on CheA activity and their in vivo behavioral effects lends convincing support to the phosphorelay model of chemotactic signaling.