Importance of Substrate Recognition and Metal Ions in the Ribonuclease P Catalysis.

[摘要] Ribonuclease P (RNase P) catalyzes the 5’ maturation of precursor-tRNA (pre-tRNA). Bacterial RNase P is composed of a catalytic RNA subunit (P RNA) and one small protein subunit (P protein); both subunits contribute to substrate recognition. Previous studies suggest that RNase P recognizes the tertiary fold of the tRNA moiety and the leader of pre-tRNA interacts with the P protein subunit. Bioinformatics analyses of 5’ leader sequences in 160 bacterial genomes demonstrated species-dependent sequence preferences near the pre-tRNA cleavage site, suggesting the formation of sequence-specific interactions with the 5’ leader. Here we demonstrate that the affinity of Bacillus subtilis RNase P for pre-tRNAAsp is modulated by the leader sequence; pre-tRNAAsp with adenosine and uracil at the N(-2) and N(-3) positions in the leader, respectively, have the highest binding affinity. Mutagenesis data indicate that a non-conserved nucleotide, G319 at J18/2 of the P RNA, forms a trans Watson-Crick-sugar-edge interaction with the base at N(-2) of pre-tRNA. Additionally, N61, R62 and R65 residues at the ;;RNR” motif of the P protein indirectly affect the N(-3) sequence selectivity.Sequence alterations at the N(-3) nucleotide also modulate the Ca2+-dependence of pre-tRNA affinity, suggesting that interactions with this nucleotide stabilize the active conformation of RNase P.The newly identified human mitochondrial (mtRNase P) and chloroplast RNase P are ;;proteinaceous” RNase P enzymes that are proposed to consist of three (MRPP1, MRPP2 and MRPP3) and one (PRORP1) protein subunits, respectively. MRPP3 is homologous to the PRORP1 protein. Sequence alignments of MRPP3 homologs indicate eight conserved residues, including cysteine, aspartate and histidine residues, located in the C-terminal domain, which have been proposed to coordinate catalytic and/or structural metal ions. Here, we demonstrate that the MRPP3 subunit is catalytically active alone in vitro, and thereby is the catalytic subunit of the human mtRNase P. Introducing the MRPP1 and MRPP2 subunits to MRPP3 subunit moderately reduces the human mtRNase P catalytic activity. Furthermore, two essential metal ions have been identified in MRPP3, including a minimum of one Mg2+ that activates catalysis and a tightly bound zinc ion that could serve to stabilize the structure of MRPP3 required for catalysis.

[发布机构] University of Michigan

[效力级别] Metal Ions

[关键词] Ribonuclease P (RNase P);Metal Ions;Substrate Recognition;Chemistry;Science;Chemical Biology