Instituto de Tecnologia Quimica e Biologica/Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal.
Biochemistry. 2009 Dec 22;48(50):11848-57. doi: 10.1021/bi901105n.
Maturation, turnover, and quality control of RNA are performed by many different classes of ribonucleases. Escherichia coli RNase II is the prototype of the RNase II family of ribonucleases, a ubiquitous family of hydrolytic, processive 3' --> 5' exonucleases crucial in RNA metabolism. RNase R is a member of this family that is modulated in response to stress and has been implicated in virulence. In this work, RNase II-like proteins were characterized in the human pathogens Salmonella typhimurium and Streptococcus pneumoniae. By sequence analysis, only one member of the RNase II family was identified in S. pneumoniae, while both RNase II and RNase R were found in Sa. typhimurium. These enzymes were cloned, expressed, purified, and characterized with regard to their biochemical features and modular architecture. The specificity of substrates and the final products generated by the enzymes were clearly demonstrated. Sa. typhimurium RNase II and RNase R behaved essentially as their respective E. coli counterparts. We have shown that the only hydrolytic RNase found in S. pneumoniae was able to degrade structured RNAs as is the case with E. coli RNase R. Our results further showed that there are differences with regard to the activity and ability to bind RNA from enzymes belonging to two distinct pneumococcal strains, and this may be related to a single amino acid substitution in the catalytic domain. Since ribonucleases have not been previously characterized in S. pneumoniae or Sa. typhimurium, this work provides an important first step in the understanding of post-transcriptional control in these pathogens.
RNA 的成熟、周转和质量控制是由许多不同类别的核糖核酸酶完成的。大肠杆菌 RNase II 是核糖核酸酶 II 家族的原型，这是一个普遍存在的水解、连续的 3' --> 5' 外切核酸酶家族，在 RNA 代谢中至关重要。RNase R 是该家族的成员，它可以响应应激而被调节，并与毒力有关。在这项工作中，在人类病原体沙门氏菌和肺炎链球菌中鉴定了 RNase II 样蛋白。通过序列分析，在肺炎链球菌中只鉴定出 RNase II 家族的一个成员，而在 Sa. typhimurium 中则发现了 RNase II 和 RNase R。这些酶被克隆、表达、纯化，并在生化特征和模块化结构方面进行了表征。清楚地证明了酶的底物特异性和最终产物。Sa. typhimurium RNase II 和 RNase R 的行为基本上与其各自的大肠杆菌对应物相同。我们已经表明，在肺炎链球菌中发现的唯一水解核糖核酸酶能够降解结构 RNA，就像大肠杆菌 RNase R 一样。我们的结果还表明，来自两个不同肺炎链球菌菌株的酶的活性和结合 RNA 的能力存在差异，这可能与催化结构域中的单个氨基酸取代有关。由于核糖核酸酶以前在肺炎链球菌或 Sa. typhimurium 中没有被表征，因此这项工作为理解这些病原体中的转录后控制提供了重要的第一步。
