Characterization of RNA aptamers binding to C5 protein, the protein component of E. coli RNase P

Abstract

RNase P is an ubiquitous endoribonuclease involved in processing of precursor tRNAs to generate the 5`` ends of mature tRNAs. RNase P derived from many species has been demonstrate to be a ribonucleoprotein complex. In Escherichia coli, the enzyme consists of a catalytic RNA subunit (M1 RNA) and a protein cofactor (C5 protein). Although M1 RNA is the catalytic subunit, C5 protein play important roles in determining substrate specificity of the RNase P reaction and in increasing the reaction rete. To understand function of C5 protein, interaction of M1 RNA and C5 protein should be examined in detail. Up to now data about interaction of M1 RNA and C5 protein is very limited mainly due to a large size of M1 RNA. One method to establish a model for interaction of protein and RNA is to analyze RNA aptamers binding to the specific protein. In this thesis, in an attempt to establish a model for interaction of M1 RNA and C5 protein, properties of RNA aptamers binding to C5 protein were characterized. The aptamers were previously selected through the 15th round by SELEX (Systematic Evolution of Ligands for Exponential enrichment) from an RNA carrying 42 bases of random sequence. The interactions between C5 protein and the RNA aptamers, M1 RNA, or precurosr tRNAs, were studied in vitro by a gel retardation assay, and the dissociation constants (Kd) for the specific interactions were determined. Dissociation constants of aptamers W2, W50, W51, and W58 are in the range of that of M1 RNA. Aptamers competed with M1 RNA for binding to C5 protein. Finally, aptamers W2 and W58 have a high binding affinity with dissociation constants of about 30nM toward precursor tRNAGlu. Possible secondary structures were proposed by a computational fold program and an enzymatic mapping. Aptamers having a higher binding affinity to C5 protein have more single-stranded region in their secondary structure.