Ribonuclease P (RNase P) catalyzes the 5' maturation of tRNAs in all three domains of life and functions as a Mg2+-dependent ribonucleoprotein (RNP) complex. It is composed of one RNA subunit, essential for catalysis, and a varying number of protein cofactors depending on the source. We have now used bacterial and archaeal RNase P to understand how proteins aid RNA catalysis.
Bacterial RNase P is composed of one catalytic RNA subunit and one protein cofactor, which is known to facilitate substrate binding and RNA catalysis. Although molecular modeling led to tertiary structure models of the RNA subunits, that were subsequently shown to be correct, and high-resolution studies established the structure of the protein subunit from bacterial RNase P, RNA-protein interactions in the holoenzyme were not established. Here, we have used a hydroxyl radical-mediated footprinting approach to generate this information which, together with results from other biochemical/biophysical studies, have furnished distance constraints for building three-dimensional models of the bacterial RNase P holoenzyme in the absence or presence of its precursor tRNA substrate. The model reveals how the protein subunit facilitates RNA catalysis by directly interacting with both the ptRNA substrate and the catalytic core of the RNA subunit.
Unlike bacterial RNase P, both archaeal and eukaryal RNase P contain multiple protein subunits, whose roles are unclear largely due to the failure to reconstitute archaeal/eukaryal RNase P in vitro. Using recombinant subunits, we have now reconstituted functional RNase P from Pyroccocus furiosus, a thermophilic archaeon, and gained insights regarding its assembly pathway(s) and the contribution of its different protein subunits to RNA catalysis. The Pfu RNase P RNA is capable of multiple turnover catalysis with either of two pairs of protein subunits and becomes significantly more active, at lower magnesium concentrations, with addition of the remaining protein pair. These data support a central tenet of the RNA world hypothesis that the evolution of RNA enzymes to RNP complexes involved gradual recruitment of proteins to enhance biological function.
Collectively, these two studies highlight the common strategies employed by protein cofactors to enhance RNA catalysis (i.e., enhanced substrate binding and improved affinity for Mg2+).
|School:||The Ohio State University|
|Department:||Molecular, Cellular, and Developmental Biology|
|School Location:||United States -- Ohio|
|Source:||DAI-B 79/09(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology|
|Keywords:||Bacterial/archaeal rnase P, RNA-protein interactions, RNP reconstitution|
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