Mutational analysis of RsrA, a zinc-binding anti-sigma factor with a thiol-disulphide redox switch
Mutational analysis of RsrA, a zinc-binding anti-sigma factor with a thiol-disulphide redox switch
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DOI：
10.1046/j.1365-2958.2001.02298.x
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发表时间：
2001-02-01
影响因子：
3.6
通讯作者：
Buttner, MJ
中科院分区：
文献类型：
--
作者：
Paget, MSB;Bae, JB;Buttner, MJ
在革兰氏阳性细菌天蓝色链霉菌A3（2）中，硫氧还蛋白系统的表达受一种名为sigma（R）的sigma因子调节，以响应细胞质硫醇二硫化物状态的变化，sigma（R）的活性在免疫后受一种反sigma因子RsrA控制。在体外，RsrA的抗σ因子活性，其中包含7个半胱氨酸，与其硫醇-二硫化物氧化还原状态。在这里，我们调查RsrA在体内的功能。构建的rsrA无效突变体具有非常高的组成型水平的二硫还原酶活性和sigma（R）依赖性转录，证实RsrA是sigma（R）的负调节因子和硫醇-二硫状态的关键传感器。靶向诱变显示，RsrA中的七个半胱氨酸中的三个（C11、C41和C44）对于抗σ因子活性是必需的，并且仅含有这三个半胱氨酸的突变体RsrA蛋白在体内是活性的并且仍然是氧化还原敏感的。我们还表明，RsrA是一种金属蛋白，含有近化学计量的锌。在这些数据的基础上，我们提出，硫醇-二硫化物氧化还原开关之间形成的C11，C41和C44的两个，所有三个残基发挥重要作用，在其还原状态下的抗σ因子活性，也许作为锌的配体。出乎意料的是，rsrA无效突变体在孢子形成中被阻断，这可能是游离σ（R）水平增加的结果。
In the Gram-positive bacterium, Streptomyces coelicolor A3(2), expression of the thioredoxin system is modulated by a sigma factor called sigma (R) in response to changes in the cytoplasmic thiol-disulphide status, and the activity of sigma (R) is controlled post-translationally by an anti-sigma factor, RsrA. In vitro, the anti-sigma factor activity of RsrA, which contains seven cysteines, correlates with its thiol-disulphide redox status. Here, we investigate the function of RsrA in vivo. A constructed rsrA null mutant had very high constitutive levels of disulphide reductase activity and sigma (R)-dependent transcription, confirming that RsrA is a negative regulator of sigma (R) and a key sensor of thiol-disulphide status. Targeted mutagenesis revealed that three of the seven cysteines in RsrA (C11, C41 and C44) were essential for anti-sigma factor activity and that a mutant RsrA protein containing only these three cysteines was active and still redox sensitive in vivo. We also show that RsrA is a metalloprotein, containing near-stoichiometric amounts of zinc. On the basis of these data, we propose that a thiol-disulphide redox switch is formed between two of C11, C41 and C44, and that all three residues play an essential role in anti-sigma factor activity in their reduced state, perhaps by acting as ligands for zinc. Unexpectedly, rsrA null mutants were blocked in sporulation, probably as a consequence of an increase in the level of free sigma (R).