The Pseudomonas aeruginosa Catabolite Repression Control Protein Crc Is Devoid of RNA Binding Activity

The Crc protein has been shown to mediate catabolite repression control in Pseudomonas, leading to a preferential assimilation of carbon sources. It has been suggested that Crc acts as a translational repressor of mRNAs, encoding functions involved in uptake and breakdown of different carbon sources. Moreover, the regulatory RNA CrcZ, the level of which is increased in the presence of less preferred carbon sources, was suggested to bind to and sequester Crc, resulting in a relief of catabolite repression. Here, we determined the crystal structure of Pseudomonas aeruginosa Crc, a member of apurinic/apyrimidinic (AP) endonuclease family, at 1.8 Å. Although Crc displays high sequence similarity with its orthologs, there are amino acid alterations in the area corresponding to the active site in AP proteins. Unlike typical AP endonuclease family proteins, Crc has a reduced overall positive charge and the conserved positively charged amino-acid residues of the DNA-binding surface of AP proteins are partially substituted by negatively charged, polar and hydrophobic residues. Crc protein purified to homogeneity from P. aeruginosa did neither display DNase activity, nor did it bind to previously identified RNA substrates. Rather, the RNA chaperone Hfq was identified as a contaminant in His-tagged Crc preparations purified by one step Ni-affinity chromatography from Escherichia coli, and was shown to account for the RNA binding activity observed with the His-Crc preparations. Taken together, these data challenge a role of Crc as a direct translational repressor in carbon catabolite repression in P. aeruginosa.

已有研究证实，Crc蛋白可介导假单胞菌（Pseudomonas）中的分解代谢物阻遏调控，使细菌优先同化碳源。此前有研究提出，Crc可作为编码不同碳源摄取与分解相关功能蛋白的信使核糖核酸（mRNA）的翻译阻遏因子。此外，调控RNA CrcZ在低偏好性碳源存在时表达水平升高，有研究认为其可结合并隔离Crc，从而解除分解代谢物阻遏。本研究解析了铜绿假单胞菌（Pseudomonas aeruginosa）Crc蛋白的晶体结构，该蛋白隶属于无嘌呤/无嘧啶（apurinic/apyrimidinic, AP）核酸内切酶家族，结构分辨率达1.8埃（Å）。尽管Crc与其直系同源物的序列相似性较高，但其对应AP蛋白活性位点的区域存在氨基酸残基变异。与典型的AP核酸内切酶家族蛋白不同，Crc的整体正电荷水平有所降低，且AP蛋白DNA结合表面保守的带正电氨基酸残基，部分被带负电、极性及疏水氨基酸残基所取代。从铜绿假单胞菌中纯化得到的均一Crc蛋白，既未表现出脱氧核糖核酸酶（DNase）活性，也无法结合此前已鉴定的RNA底物。进一步研究发现，在大肠杆菌（Escherichia coli）中通过一步镍亲和层析（Ni-affinity chromatography）纯化的组氨酸标签（His-tag）标记Crc制剂中，存在RNA分子伴侣（RNA chaperone）Hfq作为污染物，且该污染物正是导致His-Crc制剂检测到RNA结合活性的原因。综上，本研究数据对Crc在铜绿假单胞菌碳源分解代谢物阻遏过程中作为直接翻译阻遏因子的作用提出了质疑。