Department of Microbiology, University of Minnesota, Minneapolis, MN 55455.
Proc Natl Acad Sci U S A. 2014 Mar 4;111(9):3573-8. doi: 10.1073/pnas.1315374111. Epub 2014 Feb 18.
Anti-Q is a small RNA encoded on pCF10, an antibiotic resistance plasmid of Enterococcus faecalis, which negatively regulates conjugation of the plasmid. In this study we sought to understand how Anti-Q is generated relative to larger transcripts of the same operon. We found that Anti-Q folds into a branched structure that functions as a factor-independent terminator. In vitro and in vivo, termination is dependent on the integrity of this structure as well as the presence of a 3' polyuridine tract, but is not dependent on other downstream sequences. In vitro, terminated transcripts are released from RNA polymerase after synthesis. In vivo, a mutant with reduced termination efficiency demonstrated loss of tight control of conjugation function. A search of bacterial genomes revealed the presence of sequences that encode Anti-Q-like RNA structures. In vitro and in vivo experiments demonstrated that one of these functions as a terminator. This work reveals a previously unappreciated flexibility in the structure of factor-independent terminators and identifies a mechanism for generation of functional small RNAs; it should also inform annotation of bacterial sequence features, such as terminators, functional sRNAs, and operons.
抗-Q 是一种小 RNA，编码于 pCF10 上，pCF10 是粪肠球菌的一种抗生素抗性质粒，可负调控质粒的接合。在本研究中，我们试图了解抗-Q 相对于同一操纵子的较大转录本是如何产生的。我们发现抗-Q 折叠成一种具有非依赖因子终止子功能的分支结构。体外和体内实验均表明，终止取决于该结构的完整性以及 3' 多聚尿嘧啶区的存在，但不依赖于其他下游序列。体外实验表明，合成后终止转录物从 RNA 聚合酶上释放。在体内，具有降低终止效率的突变体表现出对接合功能的严格控制丧失。对细菌基因组的搜索揭示了存在编码抗-Q 样 RNA 结构的序列。体外和体内实验表明，其中一个序列作为终止子发挥作用。这项工作揭示了非依赖因子终止子结构的先前未被认识到的灵活性，并确定了功能性小 RNA 的产生机制；它还应该为细菌序列特征（如终止子、功能性 sRNA 和操纵子）的注释提供信息。
