Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci are found in bacterial and archaeal genomes where they provide the molecular machinery for acquisition of immunity against foreign DNA. In addition to the cas genes fundamentally required for CRISPR activity, a second class of genes is associated with the CRISPR loci, of which many have no reported function in CRISPR-mediated immunity. Here, we characterize MM_0565 associated to the type I-B CRISPR-locus of Methanosarcina mazei Gö1. We show that purified MM_0565 composed of a CRISPR-Cas Associated Rossmann Fold (CARF) and a winged helix-turn-helix domain forms a dimer in solution; in vivo, the dimeric MM_0565 is strongly stabilized under high salt stress. While direct effects on CRISPR-Cas transcription were not detected by genetic approaches, specific binding of MM_0565 to the leader region of both CRISPR-Cas systems was observed by microscale thermophoresis and electromobility shift assays. Moreover, overexpression of MM_0565 strongly induced transcription of the cas1-solo gene located in the recently reported casposon, the gene product of which shows high similarity to classical Cas1 proteins. Based on our findings, and taking the absence of the expressed CRISPR locus-encoded Cas1 protein into account, we hypothesize that MM_0565 might modulate the activity of the CRISPR systems on different levels.

中文翻译：

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CARF蛋白MM_0565影响马氏甲烷八叠球菌Gö1中卡斯普森编码的cas1-solo基因的转录。

在细菌和古细菌基因组中发现了成簇的规则间隔的短回文重复序列（CRISPR）基因座，它们为获得针对外源DNA的免疫力提供了分子机制。除了CRISPR活性基本需要的cas基因外，第二类基因与CRISPR基因座相关，其中许多基因在CRISPR介导的免疫中没有作用。在这里，我们表征了与马氏甲烷八叠球菌的IB CRISPR基因座相关的MM_0565Gö1。我们显示，由CRISPR-Cas相关的Rossmann折叠（CARF）和有翼的螺旋-螺旋-螺旋结构域组成的纯化的MM_0565在溶液中形成二聚体；在体内，二聚体MM_0565在高盐胁迫下具有很强的稳定性。虽然没有通过遗传方法检测到对CRISPR-Cas转录的直接影响，但通过微型热泳和电动迁移分析观察到了MM_0565与两个CRISPR-Cas系统前导区的特异性结合。此外，MM_0565的过表达强烈诱导了cas1-solo的转录该基因位于最近报道的卡斯普森，其基因产物与经典Cas1蛋白具有高度相似性。根据我们的发现，并考虑到不存在表达的CRISPR基因座编码的Cas1蛋白，我们假设MM_0565可以在不同水平上调节CRISPR系统的活性。