Lead(II) as a Tool for Probing RNA Structure in vivo

Abstract: Chemical modification and limited enzymatic hydrolysis are powerful methods to obtain detailed information on the structure and dynamics of RNAs in solution. In the work presented here I have taken advantage of the properties of the divalent metal ion lead(II) to establish it as a new probe for investigating the structure of RNA in vivo. Besides highly specific lead(II)-induced cleavage due to the presence of tight metal ion binding sites, lead(II) is known to cleave RNA within single-stranded regions, loops and bulges. The detailed structural data obtained with three different RNAs: tmRNA, CopT, and the leader region of the ompF mRNA, show that lead(II) has great potential for in vivo studies of RNA structure. In P. fluorescens, the activity and stability of RsmY, a small regulatory RNA, was shown to be strongly dependent on repeated GGA motifs in single-stranded regions. In vivo lead(II) probing essentially confirmed predicted secondary structures and also indicated binding to a protein, RsmA. The potential in using lead(II) for mapping protein binding sites on RNAs was shown for the interaction between E. coli tmRNA and the SmpB protein. In vivo and in vitro data show protections in the tRNA-like domain of tmRNA due to binding to the SmpB protein, indicating that the SmpB protein is associated with the majority of tmRNA in the cell.Furthermore, the overall conformation/ structure of E. coli RNase P was analyzed by probing the native structure of M1 RNA in vivo with lead(II). The observed cleavages suggests that M1 RNA is present in two main conformations in the cell, one being characteristic of free RNase P, and one of an RNase P-tRNA complex. The results also indicate that the C5 protein subunit has only minor effects on the overall structure of the RNA subunit.