Ribonuclease P (RNase P), the Mg2+-dependent endoribonuclease responsible for 5' end maturation of transfer RNAs (tRNAs) from their precursor transcripts, is a ribonucleoprotein (RNP) complex containing an essential RNA (RNase P RNA; RPR) and one or more protein subunits (RNase P Proteins; RPPs) in all three domains of life: one in bacteria and at least four and nine in archaea and eukarya, respectively. Since the archaeal RPPs (designated as POP5, RPP30, RPP21 and RPP29) have eukaryal homologs and archaeal RNase P is a relatively simpler RNP complex with fewer RPPs, compared to eukaryal RNase P we decided to employ archaeal RNase P as a model system to investigate the role of multiple RPPs in RNase P catalysis.
All bacterial RPRs tested thus far are catalytic (ribozymes) under certain in vitro conditions. However, despite structural similarity of their putative catalytic core to bacterial RPR, only a few representative archaeal and eukaryal RPRs are weakly active in the absence of their cognate RPPs in vitro. We reasoned that inactivity of most archaeal RPRs is likely due to structural defects that impair substrate binding. By constructing an enzyme-substrate conjugate in which a precursor tRNA substrate is tethered to the active site of Methanocaldococcus jannaschii ( Mja) RPR, which was reported to be inactive, we demonstrated that the Mja RPR alone is capable of accurately processing the ptRNA with a rate similar to that observed for the bacterial RPR.
Previous studies examining the role of RPPs in archaeal RNase P catalysis revealed that POP5-RPP30 binary complex increases the RPR's kcat to that observed with all four RPPs (POP5-RPP30 and RPP21-RPP29). Since k cat sets a lower rate limit for steps subsequent to substrate binding in the catalytic cycle, POP5-RPP30 was inferred to play a role in cleavage or product release. In this study, we assessed the effect of each binary complex on cleavage by reconstituting the ptRNA-Mja RPR conjugate with POP5-RPP30 or RPP21-RPP29 under optimal assay conditions. We found that POP5-RPP30, but not RPP21-RPP29, enhances the catalytic potential of Mja RPR by increasing both the rate of cleavage and affinity of the RPR for Mg2+. Consistent with the functional data, RNase T1 footprinting of the reconstituted Mja RNase P holoenzyme suggests that POP5-RPP30 is proximal to universally conserved nucleotides in the catalytic domain (C domain) of archaeal RPR. Moreover, a deletion derivative of archaeal RPR that retains only the C domain is able to assemble with POP5-RPP30 and generate a functional holoenzyme. Since the C domains of bacterial and archaeal RPRs are similar, we examined whether archaeal POP5-RPP30 would heterologously reconstitute with bacterial RPR. The ability to reconstitute functional holoenzymes from various evolutionarily related bacterial/archaeal/eukaryal (organellar) RPRs and archaeal RPPs revealed the presence of a conserved catalytic core in RNase P from different domains of life.
Taken together, our results provide insights into the interplay between RNA and protein subunits in RNase P catalysis and into the evolutionary transition from a simple RNP (in bacteria) to a more complex RNP (in archaea and eukarya).
|Commitee:||Alfonzo, Juan, Foster, Mark, Fredrick, Kurt, Gopalan, Venkat|
|School:||The Ohio State University|
|Department:||Ohio State Biochemistry Program|
|School Location:||United States -- Ohio|
|Source:||DAI-B 78/11(E), Dissertation Abstracts International|
|Keywords:||RNP complex, RNase P, ptRNA processing|
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