Structure and function in archaeal RNase P and the S(MK) box riboswitch

This work reports the study of relationships between structure and function in two RNA-based elements of life: archaeal RNase P and the bacterial S MK box riboswitch.;RNase P is an essential enzyme present in all three domains of life, responsible for cleaving the 5' leader sequence of precursor tRNAs, yielding mature tRNAs. The most well characterized RNase P is the bacterial version, which was shown to be an RNA enzyme, or ribozyme. Although the RNase P RNA (RPR) is the catalytic moiety, the enzyme is also a ribonucleoprotein, containing a small RNase P protein (RPP) subunit that binds to the large RNA and accounts for ∼10% of the enzyme's total mass. The RPR is catalytically active in vitro under high salt conditions, but requires the RPP for activity in vivo.;RNase P from eukaryotes is less well characterized. Its RPR is homologous to the bacterial RPR, yet no eukaryal protein has been found to share marked sequence similarity with the single bacterial RPR. Eukaryal RNase P contains at least nine protein subunits that account for ∼70% the mass of the intact enzyme and whose roles in the enzyme's function remain unknown. Remarkably, the eukaryal RPR has only nominal enzymatic activity. We hypothesize that evolution has shifted structural or catalytic responsibilities from the RPR to the RPPs in the case of the eukaryal enzyme as compared to its bacterial counterpart.;A subset of the eukaryal RPPs shares homology with four archaeal RPPs (POP5, RPP21, RPP29, and RPP30), which have been shown to enhance the activity of a cognate RPR in an in vitro reconstitution assay. Based on this observation, we adopted archaeal RNase P as a model system, with the ultimate goal of gaining insight into the complicated and poorly-understood eukaryal version of the enzyme. Archaeal RNase P is an attractive target for structural studies due to the demonstrated in vitro reconstitution, the limited number of its RPPs, the smaller size of the RPPs, and the robust solution behavior of components derived from thermophilic sources.;Work detailed here focuses primarily on the structure and interactions of archaeal RPP POP5 from the hyperthermophilic archaeon Pyrococcus furiosus. Determination of its crystal structure revealed an unexpected similarity to the bacterial RPP in spite of their different evolutionary origins. Subsequent study identified the surface that POP5 uses to interact with its partner RPP30. Finally, work was initiated to determine the structure of POP5 and RPP30 assembled with a catalytically active domain of their associated RPR.;Work performed in parallel characterized conformational changes in the SMK box riboswitch from the bacterium Enterococcus faecalis , an RNA regulatory element capable of modulating gene expression in response to fluctuating cellular concentrations of the metabolite S-adenosylmethionine. Riboswitches typically comprise two domains that communicate with each another: one for binding of the effector molecule and one for regulation of the associated gene's expression. The S MK box is of particular utility for structural characterization because its effector-binding and regulatory domains are coincident. This property allowed us to perform the first atomic-level characterization of a riboswitch interconverting between mutually exclusive, biologically pertinent conformations in response to its effector molecule.