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Differential substrate recognition by isozymes of plant protein-only Ribonuclease P.

ABSTRACT: Ribonuclease P (RNase P) catalyzes the cleavage of leader sequences from precursor tRNA (pre-tRNA). Typically, these enzymes are ribonucleic protein complexes that are found in all domains of life. However, a new class of RNase P has been discovered that is composed entirely of protein, termed protein-only RNase P (PRORP). To investigate the molecular determinants of PRORP substrate recognition, we measured the binding affinities and cleavage kinetics of Arabidopsis PRORP1 for varied pre-tRNA substrates. This analysis revealed that PRORP1 does not make significant contacts within the trailer or beyond N-1of the leader, indicating that this enzyme recognizes primarily the tRNA body. To determine the extent to which sequence variation within the tRNA body modulates substrate selectivity and to provide insight into the evolution and function of PRORP enzymes, we measured the reactivity of the three Arabidopsis PRORP isozymes (PRORP1-3) with four pre-tRNA substrates. A 13-fold range in catalytic efficiencies (10(4)-10(5)M(-1)s(-1)) was observed, demonstrating moderate selectivity for pre-tRNA substrates. Although PRORPs bind the different pre-tRNA species with affinities varying by as much as 100-fold, the three isozymes have similar affinities for a given pre-tRNA, suggesting similar binding modes. However, PRORP isozymes have varying degrees of cleavage fidelity, which is dependent on the pre-tRNA species and the presence of a 3'-discriminator base. This work defines molecular determinants of PRORP substrate recognition that provides insight into this new class of RNA processing enzymes.

SUBMITTER: Howard MJ

PROVIDER: S-EPMC4836652 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Differential substrate recognition by isozymes of plant protein-only Ribonuclease P.

Howard Michael J MJ Karasik Agnes A Klemm Bradley P BP Mei Christine C Shanmuganathan Aranganathan A Fierke Carol A CA Koutmos Markos M

RNA (New York, N.Y.) 20160310 5

Ribonuclease P (RNase P) catalyzes the cleavage of leader sequences from precursor tRNA (pre-tRNA). Typically, these enzymes are ribonucleic protein complexes that are found in all domains of life. However, a new class of RNase P has been discovered that is composed entirely of protein, termed protein-only RNase P (PRORP). To investigate the molecular determinants of PRORP substrate recognition, we measured the binding affinities and cleavage kinetics of Arabidopsis PRORP1 for varied pre-tRNA su ...[more]

PMID: 26966150

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Project description:Bacterial ribonuclease P (RNase P) catalyzes the cleavage of 5' leader sequences from precursor tRNAs (pre-tRNAs). Previously, all known substrate nucleotide specificities in this system are derived from RNA-RNA interactions with the RNase P RNA subunit. Here, we demonstrate that pre-tRNA binding affinities for Bacillus subtilis and Escherichia coli RNase P are enhanced by sequence-specific contacts between the fourth pre-tRNA nucleotide on the 5' side of the cleavage site (N(-4)) and the RNase P protein (P protein) subunit. B. subtilis RNase P has a higher affinity for pre-tRNA with adenosine at N(-4), and this binding preference is amplified at physiological divalent ion concentrations. Measurements of pre-tRNA-containing adenosine analogs at N(-4) indicate that specificity arises from a combination of hydrogen bonding to the N6 exocyclic amine of adenosine and steric exclusion of the N2 amine of guanosine. Mutagenesis of B. subtilis P protein indicates that F20 and Y34 contribute to selectivity at N(-4). The hydroxyl group of Y34 enhances selectivity, likely by forming a hydrogen bond with the N(-4) nucleotide. The sequence preference of E. coli RNase P is diminished, showing a weak preference for adenosine and cytosine at N(-4), consistent with the substitution of Leu for Y34 in the E. coli P protein. This is the first identification of a sequence-specific contact between P protein and pre-tRNA that contributes to molecular recognition of RNase P. Additionally, sequence analyses reveal that a greater-than-expected fraction of pre-tRNAs from both E. coli and B. subtilis contains a nucleotide at N(-4) that enhances RNase P affinity. This observation suggests that specificity at N(-4) contributes to substrate recognition in vivo. Furthermore, bioinformatic analyses suggest that sequence-specific contacts between the protein subunit and the leader sequences of pre-tRNAs may be common in bacterial RNase P and may lead to species-specific substrate recognition.

Dataset Information

Differential substrate recognition by isozymes of plant protein-only Ribonuclease P.

Publications

Differential substrate recognition by isozymes of plant protein-only Ribonuclease P.

Similar Datasets

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