Structural insight into precursor tRNA processing by yeast ribonuclease P

Science ◽  
2018 ◽  
Vol 362 (6415) ◽  
pp. eaat6678 ◽  
Author(s):  
Pengfei Lan ◽  
Ming Tan ◽  
Yuebin Zhang ◽  
Shuangshuang Niu ◽  
Juan Chen ◽  
...  
Keyword(s):  
Metal Ion ◽  
Simulation Analysis ◽  
Reaction Pathway ◽  
Rnase P ◽  
Ribonuclease P ◽  
Measuring Device ◽  
Catalytic Center ◽  
Cryo Electron Microscopy ◽  
Phosphate Backbone ◽  
Protein Components

Ribonuclease P (RNase P) is a universal ribozyme responsible for processing the 5′-leader of pre–transfer RNA (pre-tRNA). Here, we report the 3.5-angstrom cryo–electron microscopy structures of Saccharomyces cerevisiae RNase P alone and in complex with pre-tRNAPhe. The protein components form a hook-shaped architecture that wraps around the RNA and stabilizes RNase P into a “measuring device” with two fixed anchors that recognize the L-shaped pre-tRNA. A universally conserved uridine nucleobase and phosphate backbone in the catalytic center together with the scissile phosphate and the O3′ leaving group of pre-tRNA jointly coordinate two catalytic magnesium ions. Binding of pre-tRNA induces a conformational change in the catalytic center that is required for catalysis. Moreover, simulation analysis suggests a two-metal-ion SN2 reaction pathway of pre-tRNA cleavage. These results not only reveal the architecture of yeast RNase P but also provide a molecular basis of how the 5′-leader of pre-tRNA is processed by eukaryotic RNase P.

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

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.

scholarly journals Cryo-electron microscopy structure of an archaeal ribonuclease P holoenzyme

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Futang Wan ◽  
Qianmin Wang ◽  
Jing Tan ◽  
Ming Tan ◽  
Juan Chen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ribonuclease P ◽  
Cryo Electron Microscopy

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

2019 ◽  
Vol 47 (12) ◽  
pp. 6425-6438 ◽  
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.

Simulation analysis of structures on the reaction pathway of RNAse A

1990 ◽  
Vol 112 (10) ◽  
pp. 3826-3831 ◽  
Author(s):  
Karen Haydock ◽  
Carmay Lim ◽  
Axel T. Brunger ◽  
Martin Karplus
Keyword(s):  
Simulation Analysis ◽  
Reaction Pathway ◽  
Rnase A

scholarly journals Ribonuclease P

2011 ◽  
Vol 366 (1580) ◽  
pp. 2936-2941 ◽  
Author(s):  
Sidney Altman
Keyword(s):  
Rnase P ◽  
Evolutionary Tree ◽  
Ancient World ◽  
Ribonuclease P ◽  
Free Living ◽  
Rna Molecules ◽  
Gene Coding ◽  
Living Organisms

The gene coding for the RNA subunit of ribonuclease P (RNase P) is essential in all free-living organisms. The RNA subunit, itself, is an enzyme and, from its evolutionary tree, we can infer that it is a very ancient molecule. The specificity of this enzyme is that it cleaves other RNA molecules at the junction of single-stranded and the 5′ end of double-stranded regions of RNA. One can speculate that this molecule was very useful in an ancient world in cleaving long pieces of RNA, which must have contained hairpin regions in it, into shorter molecules with the capability of different functions from the longer parent. Today, the specificity of the enzyme can be used in designing drug therapies.

Characterizations of Nanostructured Films as Responsive Electrode Materials

2001 ◽  
Vol 704 ◽  
Author(s):  
Nancy Kariuki ◽  
Jin Luo ◽  
Laura Moussa ◽  
Lisa B. Israel ◽  
Chuan-Jian Zhong ◽  
...  
Keyword(s):  
Metal Ion ◽  
Electrode Materials ◽  
Reaction Pathway ◽  
Ionic Species ◽  
Precipitation Reaction ◽  
Void Space ◽  
Space Forms ◽  
Responsive Materials ◽  
Mass Fluxes ◽  
Carboxylate Groups

AbstractNanostructured thin films were assembled as metal-responsive electrode materials from monolayer-capped gold nanoparticles (2 nm) and carboxylic acid functionalized alkyl thiol linkers via an exchange-crosslinking-precipitation reaction pathway. The network assemblies have open frameworks in which void space forms channels or chambers with the nanometer sized cores defining its size and the shell structures defining its chemical specificity. Such nanostructures were investigated as responsive materials for the detection of metal ion fluxes. Cyclic voltammetry, in-situ electrochemical quartz-crystal nanobalance, and surface infrared reflection spectroscopy techniques were used to characterize the interfacial redox reactivity and mass fluxes at the nanostructured electrode materials. The system showed remarkable reversible mass loading arising from incorporation of ionic species into the film. The diagnostic stretching bands of the carboxylic and carboxylate groups at the shell allowed the identification and assessment of the interfacial carboxylate-metal ion reactivity.

scholarly journals Suitability and Sufficiency of Telehealth Clinician-Observed, Participant-Collected Samples for SARS-CoV-2 Testing: The iCollect Cohort Pilot Study

10.2196/19731 ◽  
2020 ◽  
Vol 6 (2) ◽  
pp. e19731 ◽  
Author(s):  
Jodie L Guest ◽  
Patrick S Sullivan ◽  
Mariah Valentine-Graves ◽  
Rachel Valencia ◽  
Elizabeth Adam ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rnase P ◽  
Ribonuclease P ◽  
Laboratory Assessment ◽  
Testing Strategies ◽  
Specimen Collection ◽  
Oropharyngeal Swab ◽  
Polymerase Chain ◽  
Respiratory Coronavirus ◽  
Serology Testing

Background The severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic calls for expanded opportunities for testing, including novel testing strategies such as home-collected specimens. Objective We aimed to understand whether oropharyngeal swab (OPS), saliva, and dried blood spot (DBS) specimens collected by participants at home and mailed to a laboratory were sufficient for use in diagnostic and serology tests of SARS-CoV-2. Methods Eligible participants consented online and were mailed a participant-collection kit to support collection of three specimens for SARS-CoV-2 testing: saliva, OPS, and DBS. Participants performed the specimen collection procedures during a telehealth video appointment while clinical observers watched and documented the suitability of the collection. The biological sufficiency of the specimens for detection of SARS-CoV-2 by reverse transcriptase–polymerase chain reaction and serology testing was assessed by laboratorians using visual inspection and quantification of the nucleic acid contents of the samples by ribonuclease P (RNase P) measurements. Results Of the enrolled participants,153/159 (96.2%) returned their kits, which were included in this analysis. All these participants attended their video appointments. Clinical observers assessed that of the samples collected, 147/153 (96.1%) of the saliva samples, 146/151 (96.7%) of the oropharyngeal samples, and 135/145 (93.1%) of the DBS samples were of sufficient quality for submission for laboratory testing; 100% of the OPS samples and 98% of the saliva samples had cycle threshold values for RNase P <30, indicating that the samples contained sufficient nucleic acid for RNA-PCR testing for SARS-CoV-2. Conclusions These pilot data indicate that most participant-collected OPS, saliva, and DBS specimens are suitable and sufficient for testing for SARS-CoV-2 RNA and serology. Clinical observers rated the collection of specimens as suitable for testing, and visual and quantitative laboratory assessment indicated that the specimens were biologically sufficient. These data support the utility of participant-collected and mailed-in specimens for SARS-CoV-2 testing. International Registered Report Identifier (IRRID) RR2-10.2196/19054

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