Crystal Structure of a bacterial RNase P holoenzyme in complex with tRNA and in the presence of 5' leader
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Ribonuclease P (RNase P) is an endoribonuclease that catalyzes the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). Ribonucleoprotein RNase P and protein-only RNase P (PRORP) in eukaryotes have been extensively studied, but the mechanism by which a prokaryotic nuclease recognizes and cleaves pre-tRNA is unclear. To gain insights into this mechanism, we studied homologs of Aquifex RNase P (HARPs), thought to be enzymes of approximately 23 kDa comprising only this nuclease domain. We determined the cryo-EM structure of Aq880, the first identified HARP enzyme. The structure unexpectedly revealed that Aq880 consists of both the nuclease and protruding helical (PrH) domains. Aq880 monomers assemble into a dimer via the PrH domain. Six dimers form a dodecamer with a left-handed one-turn superhelical structure. The structure also revealed that the active site of Aq880 is analogous to that of eukaryotic PRORPs. The pre-tRNA docking model demonstrated that 5' processing of pre-tRNAs is achieved by two adjacent dimers within the dodecamer. One dimer is responsible for catalysis, and the PrH domains of the other dimer are responsible for pre-tRNA elbow recognition. Our study suggests that HARPs measure an invariant distance from the pre-tRNA elbow to cleave the 5' leader sequence, which is analogous to the mechanism of eukaryotic PRORPs and the ribonucleoprotein RNase P. Collectively, these findings shed light on how different types of RNase P enzymes utilize the same pre-tRNA processing.
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Ribonuclease P (RNase P) catalyzes removal of 5′ end leader sequences from precursor tRNA (pre-tRNA). In most organisms, RNase P is a ribonucleoprotein complex with a catalytic RNA subunit. However, a recently discovered human mitochondrial RNase P (mtRNase P) is composed solely of three protein subunits: MRPP1, 2 and 3. The MRPP1•MRPP2 subcomplex contains m1N9 tRNA methyltransferase (MRPP1), while MRPP3 belongs to a new class of metallonucleases. We reconstituted human mtRNase P proteins in vitro and measured reactivity with different types of pre-tRNA substrate to study substrate recognition. For a canonical pre-tRNA, transient kinetic measurements show that the MRPP1•MRPP2 subcomplex activates the endonuclease activity of MRPP3 by about 2000-fold and enhances substrate affinity by 50-fold. For a non-canonical human mt-pre-tRNA, significant miscleavage was observed with MRPP3 alone but the MRPP1•MRPP2 subcomplex rescued miscleavage and enhanced the cleavage rate constant by 1000-fold. In addition, in vitro pull-down results indicate that MRPP3 mainly interacts with the (MRPP1•MRPP2)•pre-tRNA complex. These data suggest that MRPP3 recognizes the MRPP1•MRPP2-bound pre-tRNA as substrate. The unusual structural features of human mitochondrial tRNA may have been part of the evolutionary driving force for this unique strategy for substrate recognition. We are carrying out further investigations into the molecular details of this mechanism using transient and steady-state kinetics, structure probing, and mass spectrometry. [(This work is supported by funding from NIH (GM 55387)]
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The 5'-terminal guanylate residue (G-1) of mature Escherichia coli tRNA(His) is generated as a result of an unusual cleavage by RNase P (Orellana, O., Cooley, L., and Soll, D. (1986) Mol. Cell. Biol. 6, 525-529). We have examined the importance of the unique acceptor stem structure of E. coli tRNA(His) in determining the specificity of RNase P cleavage. Mutant tRNA(His) precursors bearing substitutions of the normal base G-1 or the opposing, potentially paired base, C73, can be cleaved at the +1 position, in contrast to wild-type precursors which are cut exclusively at the -1 position. These data indicate that the nature of the base at position -1 is of greater importance in determining the site of RNase P cleavage than potential base pairing between nucleotides -1 and 73. In addition, processing of the mutant precursors by M1-RNA or P RNA under conditions of ribozyme catalysis yields a higher proportion of +1-cleaved products in comparison to the reaction catalyzed by the RNase P holoenzyme. This lower sensitivity of the holoenzyme to alterations in acceptor stem structure suggests that the protein moiety of RNase P may play a role in determining the specificity of the reaction and implies that recognition of the substrate involves additional regions of the tRNA. We have also shown that the RNase P holoenzyme and tRNA(His) precursor of Saccharomyces cerevisiae, unlike their prokaryotic counterparts, do not possess these abilities to carry out this unusual reaction.
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