Probing RNA structures with hydroxyl radicals.

Fe(II)-EDTA can be used to conveniently generate hydroxyl radicals to promote cleavage of RNA at nucleotide resolution. Two procedures are described, involving the generation of free radicals from solvated molecular oxygen and from hydrogen peroxide added to the RNA solution. Unlike other footprinting reagents, hydroxyl radicals cleave the sugar-phosphate backbone at every residue and thus provide uniform cleavage in a given RNA secondary structure.

Novel RNA-binding properties of Pop3p support a role for eukaryotic RNase P protein subunits in substrate recognition.

Ribonuclease P (RNase P) catalyzes the 5'-end maturation of transfer RNA molecules. Recent evidence suggests that the eukaryotic protein subunits may provide substrate-binding functions (True, H. L.

Translationally repressive RNA structures monitored in vivo using temperate DNA bacteriophages.

The RNA challenge phage system enables genetic selection of proteins with RNA-binding activity in bacteria. These phages are modified versions of the temperate DNA bacteriophage P22 in which post-transcriptional regulatory events control the developmental fate of the phage. The system was originally developed to identify novel RNA ligands that display reduced affinity for the R17/MS2 coat protein, as well as to select for suppressor coat proteins that recognize mutant RNA ligands.

Protein components contribute to active site architecture for eukaryotic ribonuclease P.

In eukaryotes, ribonuclease P (RNase P) requires both RNA and protein components for catalytic activity. The eukaryotic RNase P RNA, unlike its bacterial counterparts, does not possess intrinsic catalytic activity in the absence of holoenzyme protein components. We have used a sensitive photoreactive cross-linking assay to explore the substrate-binding environment for different eukaryotic RNase P holoenzymes.