Ribonuclease P Protein Subunit p40

Staphylococcus aureus ribonuclease-P-protein subunit (RnpA) is a promising antimicrobial target that is a key protein component for two essential cellular processes, RNA degradation and transfer-RNA (tRNA) maturation. The first crystal structure of RnpA from the pathogenic bacterial species, S. aureus, is reported at 2.0 Å resolution. The structure presented maintains key similarities with previously reported RnpA structures from bacteria and archaea, including the highly conserved RNR-box region and aromatic residues in the precursor-tRNA 5′-leader-binding domain. This structure will be instrumental in the pursuit of structure-based designed inhibitors targeting RnpA-mediated RNA processing as a novel therapeutic approach for treating S. aureus infections.

Download Full-text

Interplay between substrate recognition, 5’ end tRNA processing and methylation activity of human mitochondrial RNase P

10.1101/452920 ◽

2018 ◽

Author(s):

Agnes Karasik ◽

Carol A. Fierke ◽

Markos Koutmos

Keyword(s):

Mitochondrial Diseases ◽

Protein S ◽

Rnase P ◽

Ribonuclease P ◽

Mitochondrial Rna ◽

P Protein ◽

Adenosyl Methionine ◽

Methylation Activity

ABSTRACTHuman mitochondrial ribonuclease P (mtRNase P) is an essential three protein complex that catalyzes the 5’ end maturation of mitochondrial precursor tRNAs (pre-tRNAs). MRPP3 (Mitochondrial RNase P Protein 3), a protein-only RNase P (PRORP), is the nuclease component of the mtRNase P complex and requires a two-protein S-adenosyl methionine (SAM)-dependent methyltransferase MRPP1/2 sub-complex to function. Dysfunction of mtRNase P is linked to several human mitochondrial diseases, such as mitochondrial myopathies. Despite its central role in mitochondrial RNA processing, little is known about how the protein subunits of mtRNase P function synergistically. Here we use purified mtRNase P to demonstrate that mtRNase P recognizes, cleaves, and methylates some, but not all, mitochondrial pre-tRNAs in vitro. Additionally, mtRNase P does not process all mitochondrial pre-tRNAs uniformly, suggesting the possibility that some pre-tRNAs require additional factors to be cleaved in vivo. Consistent with this, we found that addition of the MRPP1 co-factor SAM enhances the ability of mtRNase P to bind and cleave some mitochondrial pre-tRNAs. Furthermore, the presence of MRPP3 can enhance the methylation activity of MRPP1/2. Taken together, our data demonstrate that the subunits of mtRNase P work together to efficiently recognize, process and methylate human mitochondrial pre-tRNAs.

Download Full-text

Fluorescence properties of a tryptophan residue in an aromatic core of the protein subunit of ribonuclease P from Escherichia coli

Journal of Molecular Biology ◽

10.1006/jmbi.1997.0907 ◽

1997 ◽

Vol 267 (4) ◽

pp. 765-769 ◽

Cited By ~ 15

Author(s):

Venkat Gopalan ◽

Ralph Golbik ◽

Gideon Schreiber ◽

Alan R. Fersht ◽

Sidney Altman

Keyword(s):

Escherichia Coli ◽

Fluorescence Properties ◽

Ribonuclease P ◽

Tryptophan Residue ◽

Protein Subunit

Download Full-text

A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor

Nucleic Acids Research ◽

10.1093/nar/gkz285 ◽

2019 ◽

Vol 47 (12) ◽

pp. 6425-6438 ◽

Cited By ~ 1

Author(s):

Ezequiel-Alejandro Madrigal-Carrillo ◽

Carlos-Alejandro Díaz-Tufinio ◽

Hugo-Aníbal Santamaría-Suárez ◽

Marcelino Arciniega ◽

Alfredo Torres-Larios

Keyword(s):

Rna Processing ◽

Rnase P ◽

Ribonuclease P ◽

Binding Assays ◽

Antibiotic Development ◽

Processing Enzymes ◽

P Protein ◽

Molecule Inhibitor ◽

Screening Platform ◽

Dynamics Simulations

AbstractRibonucleoprotein (RNP) complexes and RNA-processing enzymes are attractive targets for antibiotic development owing to their central roles in microbial physiology. For many of these complexes, comprehensive strategies to identify inhibitors are either lacking or suffer from substantial technical limitations. Here, we describe an activity-binding-structure platform for bacterial ribonuclease P (RNase P), an essential RNP ribozyme involved in 5′ tRNA processing. A novel, real-time fluorescence-based assay was used to monitor RNase P activity and rapidly identify inhibitors using a mini-helix and a pre-tRNA-like bipartite substrate. Using the mini-helix substrate, we screened a library comprising 2560 compounds. Initial hits were then validated using pre-tRNA and the pre-tRNA-like substrate, which ultimately verified four compounds as inhibitors. Biolayer interferometry-based binding assays and molecular dynamics simulations were then used to characterize the interactions between each validated inhibitor and the P protein, P RNA and pre-tRNA. X-ray crystallographic studies subsequently elucidated the structure of the P protein bound to the most promising hit, purpurin, and revealed how this inhibitor adversely affects tRNA 5′ leader binding. This integrated platform affords improved structure-function studies of RNA processing enzymes and facilitates the discovery of novel regulators or inhibitors.

Download Full-text

Cloning, Purification and Characterization of the Protein Subunit of Ribonuclease P from the Cyanobacterium Synechocystis sp. PCC 6803

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1996.0017t.x ◽

1996 ◽

Vol 241 (1) ◽

pp. 17-24 ◽

Cited By ~ 15

Author(s):

Alberto Pascual ◽

Agustin Vioque

Keyword(s):

Ribonuclease P ◽

Protein Subunit ◽

Purification And Characterization ◽

Synechocystis Sp ◽

Pcc 6803

Download Full-text

Yeast mitochondrial RNase P RNA synthesis is altered in an RNase P protein subunit mutant: insights into the biogenesis of a mitochondrial RNA-processing enzyme.

Molecular and Cellular Biology ◽

10.1128/mcb.16.7.3429 ◽

1996 ◽

Vol 16 (7) ◽

pp. 3429-3436 ◽

Cited By ~ 16

Author(s):

V Stribinskis ◽

G J Gao ◽

P Sulo ◽

Y L Dang ◽

N C Martin

Keyword(s):

Rna Processing ◽

Mitochondrial Protein ◽

Rnase P ◽

Mitochondrial Rna ◽

Wild Type ◽

Protein Subunit ◽

Mitochondrial Trna ◽

P Deficiency ◽

F Gene ◽

P Protein

Rpm2p is a protein subunit of Saccharomyces cerevisiae yeast mitochondrial RNase P, an enzyme which removes 5' leader sequences from mitochondrial tRNA precursors. Precursor tRNAs accumulate in strains carrying a disrupted allele of RPM2. The resulting defect in mitochondrial protein synthesis causes petite mutants to form. We report here that alteration in the biogenesis of Rpm1r, the RNase P RNA subunit, is another consequence of disrupting RPM2. High-molecular-weight transcripts accumulate, and no mature Rpm1r is produced. Transcript mapping reveals that the smallest RNA accumulated is extended on both the 5' and 3' ends relative to mature Rpm1r. This intermediate and other longer transcripts which accumulate are also found as low-abundance RNAs in wild-type cells, allowing identification of processing events necessary for conversion of the primary transcript to final products. Our data demonstrate directly that Rpm1r is transcribed with its substrates, tRNA met f and tRNAPro, from a promoter located upstream of the tRNA met f gene and suggest that a portion also originates from a second promoter, located between the tRNA met f gene and RPM1. We tested the possibility that precursors accumulate because the RNase P deficiency prevents the removal of the downstream tRNAPro. Large RPM1 transcripts still accumulate in strains missing this tRNA. Thus, an inability to process cotranscribed tRNAs does not explain the precursor accumulation phenotype. Furthermore, strains with mutant RPM1 genes also accumulate precursor Rpm1r, suggesting that mutations in either gene can lead to similar biogenesis defects. Several models to explain precursor accumulation are presented.

Download Full-text

RNase P protein subunit Rpp29 represses histone H3.3 nucleosome deposition

Molecular Biology of the Cell ◽

10.1091/mbc.e15-02-0099 ◽

2016 ◽

Vol 27 (7) ◽

pp. 1154-1169 ◽

Cited By ~ 10

Author(s):

Alyshia Newhart ◽

Sara Lawrence Powers ◽

Prashanth Krishna Shastrula ◽

Isabel Sierra ◽

Lucy M. Joo ◽

...

Keyword(s):

Chromatin Remodeling ◽

Antisense Rna ◽

Rnase P ◽

Protein Subunit ◽

P Protein ◽

Histone H3.3 ◽

Single Living Cells ◽

Rna Complex ◽

Nucleolar Rna ◽

Single Living

In mammals, histone H3.3 is a critical regulator of transcription state change and heritability at both euchromatin and heterochromatin. The H3.3-specific chaperone, DAXX, together with the chromatin-remodeling factor, ATRX, regulates H3.3 deposition and transcriptional silencing at repetitive DNA, including pericentromeres and telomeres. However, the events that precede H3.3 nucleosome incorporation have not been fully elucidated. We previously showed that the DAXX-ATRX-H3.3 pathway regulates a multi-copy array of an inducible transgene that can be visualized in single living cells. When this pathway is impaired, the array can be robustly activated. H3.3 is strongly recruited to the site during activation where it accumulates in a complex with transcribed sense and antisense RNA, which is distinct from the DNA/chromatin. This suggests that transcriptional events regulate H3.3 recruited to its incorporation sites. Here we report that the nucleolar RNA proteins Rpp29, fibrillarin, and RPL23a are also components of this H3.3/RNA complex. Rpp29 is a protein subunit of RNase P. Of the other subunits, POP1 and Rpp21 are similarly recruited suggesting that a variant of RNase P regulates H3.3 chromatin assembly. Rpp29 knockdown increases H3.3 chromatin incorporation, which suggests that Rpp29 represses H3.3 nucleosome deposition, a finding with implications for epigenetic regulation.

Download Full-text