Structure-activity relationship and thermostability of ribonucleoprotein enzyme

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The ribonuclease P (RNase P) in the hyperthermophilic archaeon Pyrococcus horikoshiicomprises RNA (PhopRNA) and five proteins. We analyzed the RNA binding mode of the proteins, using a pair of complementary fluorescence-labeled oligoribonucleotides. Fluorescence resonance energy transfer (FRET)-based assayssuggests that the RNase P proteins assist PhopRNA in attaining a functionally active conformation via a distinct mode of binding. Moreover, Thermodynamic analysis revealed that PhoRpp38 and PhopRNA interact with each other with an association constant (Ka) of 1.56 x 107M-1. It was further found that PhoRpp38 simultaneously binds two stem-loop structures in PhopRNA approximately with an equal affinity. On the basis of the results, it was suggested that the simultaneous binding of PhoRpp38 to SL1 and SL2 makes an appropriate orientation of the S and C domains and that PhoRpp38 binding stabilizes base pairing, which increases base staking in PhopRNA