Interaction of C5 protein with RNA aptamers selected by SELEX

Abstract

Escherichia coli RNase P, a tRNA processing enzyme, is composed of M1 RNA and C5 protein. While the role of M1 RNA as the catalytic subunit has been well documented, understanding of the functions of C5 protein as a RNA-binding protein in vivo remains very limited. Interactions of proteins with RNA are important to gene expression and several proteins that bind to RNA are known to be involved in gene regulation. Although C5 protein was originally identified as the protein component of RNase P, it is possible that C5 protein participates in other cellular metabolisms. to understand the function of C5 protein as an RNA binding protein, RNA aptamers binding to C5 protein were selected by SELEX (Systematic Evolution of Ligands by EXponential enrichment) from a library of 108-mers that contained a central variable segment of 42 nucleotides. Sequence analysis of the RNA aptamers suggests that C5 protein can bind to various RNA motifs with comparable dissociation constant to that of M1 RNA. The predominant sequence, named W2, was selected for further study. Interaction between W2 and C5 protein was independent of the $Mg^{2+}$ concentration, which contrasts with the $Mg^{2+}$ -dependency of the M1 RNA-C5 protein interaction. The stability of the ribonucleoprotein of W2-C5 protein was much lower than that of M1 RNA, but it increased with lowering temperatures. The affinity of W2 to C5 protein increased with the concentration of monovalent ion $NH_4^{+}$, suggesting that the interaction occurs through hydrophobic attractions. A core RNA motif essential for interaction with C5 protein was identified as a stem-loop structure composed of the 5-bp stem and 20-nt loop. Many RNA motifs similar to the core RNA motif were present in the E. coli genome sequence. This may imply that C5 protein can function as an interacting partner protein of other cellular RNAs than M1 RNA in in vivo. To identify RNA motifs interacting with C5 protein in vivo, genomic SELEX was carried out with...