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Bacterial RNA-free RNase P: Structural and functional characterization of multiple oligomeric forms of a minimal protein-only ribonuclease P

tRNAs are typically transcribed with extended 5′ and 3′ ends that must be removed before they attain their active form. One of the first steps of tRNA processing in nearly every organism is the removal of the 5′ leader sequence by ribonuclease P (RNase P). Here, we investigate a recently discovered...

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Autores principales: Wilhelm, Catherine A., Mallik, Leena, Kelly, Abigail L., Brotzman, Shayna, Mendoza, Johnny, Anders, Anna G., Leskaj, Suada, Castillo, Carmen, Ruotolo, Brandon T., Cianfrocco, Michael A., Koutmos, Markos
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10652100/
https://www.ncbi.nlm.nih.gov/pubmed/37806495
http://dx.doi.org/10.1016/j.jbc.2023.105327
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author Wilhelm, Catherine A.
Mallik, Leena
Kelly, Abigail L.
Brotzman, Shayna
Mendoza, Johnny
Anders, Anna G.
Leskaj, Suada
Castillo, Carmen
Ruotolo, Brandon T.
Cianfrocco, Michael A.
Koutmos, Markos
author_facet Wilhelm, Catherine A.
Mallik, Leena
Kelly, Abigail L.
Brotzman, Shayna
Mendoza, Johnny
Anders, Anna G.
Leskaj, Suada
Castillo, Carmen
Ruotolo, Brandon T.
Cianfrocco, Michael A.
Koutmos, Markos
author_sort Wilhelm, Catherine A.
collection PubMed
description tRNAs are typically transcribed with extended 5′ and 3′ ends that must be removed before they attain their active form. One of the first steps of tRNA processing in nearly every organism is the removal of the 5′ leader sequence by ribonuclease P (RNase P). Here, we investigate a recently discovered class of RNase P enzymes, Homologs of Aquifex RNase P (HARPs). In contrast to other RNase Ps, HARPs consist only of a metallonuclease domain and lack the canonical substrate recognition domain essential in other classes of proteinaceous RNase P. We determined the cryo-EM structure of Aquifex aeolicus HARP (Aq880) and two crystal structures of Hydrogenobacter thermophilus HARP (Hth1307) to reveal that both enzymes form large ring-like assemblies: a dodecamer in Aq880 and a tetradecamer in Hth1307. In both oligomers, the enzyme active site is 42 Å away from a positively charged helical region, as seen in other protein-only RNase P enzymes, which likely serves to recognize and bind the elbow region of the pre-tRNA substrate. In addition, we use native mass spectrometry to confirm and characterize the previously unreported tetradecamer state. Notably, we find that multiple oligomeric states of Hth1307 are able to cleave pre-tRNAs. Furthermore, our single-turnover kinetic studies indicate that Hth1307 cleaves pre-tRNAs from multiple species with a preference for native substrates. These data provide a closer look at the nuanced similarities and differences in tRNA processing across disparate classes of RNase P.
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spelling pubmed-106521002023-10-06 Bacterial RNA-free RNase P: Structural and functional characterization of multiple oligomeric forms of a minimal protein-only ribonuclease P Wilhelm, Catherine A. Mallik, Leena Kelly, Abigail L. Brotzman, Shayna Mendoza, Johnny Anders, Anna G. Leskaj, Suada Castillo, Carmen Ruotolo, Brandon T. Cianfrocco, Michael A. Koutmos, Markos J Biol Chem Research Article tRNAs are typically transcribed with extended 5′ and 3′ ends that must be removed before they attain their active form. One of the first steps of tRNA processing in nearly every organism is the removal of the 5′ leader sequence by ribonuclease P (RNase P). Here, we investigate a recently discovered class of RNase P enzymes, Homologs of Aquifex RNase P (HARPs). In contrast to other RNase Ps, HARPs consist only of a metallonuclease domain and lack the canonical substrate recognition domain essential in other classes of proteinaceous RNase P. We determined the cryo-EM structure of Aquifex aeolicus HARP (Aq880) and two crystal structures of Hydrogenobacter thermophilus HARP (Hth1307) to reveal that both enzymes form large ring-like assemblies: a dodecamer in Aq880 and a tetradecamer in Hth1307. In both oligomers, the enzyme active site is 42 Å away from a positively charged helical region, as seen in other protein-only RNase P enzymes, which likely serves to recognize and bind the elbow region of the pre-tRNA substrate. In addition, we use native mass spectrometry to confirm and characterize the previously unreported tetradecamer state. Notably, we find that multiple oligomeric states of Hth1307 are able to cleave pre-tRNAs. Furthermore, our single-turnover kinetic studies indicate that Hth1307 cleaves pre-tRNAs from multiple species with a preference for native substrates. These data provide a closer look at the nuanced similarities and differences in tRNA processing across disparate classes of RNase P. American Society for Biochemistry and Molecular Biology 2023-10-06 /pmc/articles/PMC10652100/ /pubmed/37806495 http://dx.doi.org/10.1016/j.jbc.2023.105327 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Wilhelm, Catherine A.
Mallik, Leena
Kelly, Abigail L.
Brotzman, Shayna
Mendoza, Johnny
Anders, Anna G.
Leskaj, Suada
Castillo, Carmen
Ruotolo, Brandon T.
Cianfrocco, Michael A.
Koutmos, Markos
Bacterial RNA-free RNase P: Structural and functional characterization of multiple oligomeric forms of a minimal protein-only ribonuclease P
title Bacterial RNA-free RNase P: Structural and functional characterization of multiple oligomeric forms of a minimal protein-only ribonuclease P
title_full Bacterial RNA-free RNase P: Structural and functional characterization of multiple oligomeric forms of a minimal protein-only ribonuclease P
title_fullStr Bacterial RNA-free RNase P: Structural and functional characterization of multiple oligomeric forms of a minimal protein-only ribonuclease P
title_full_unstemmed Bacterial RNA-free RNase P: Structural and functional characterization of multiple oligomeric forms of a minimal protein-only ribonuclease P
title_short Bacterial RNA-free RNase P: Structural and functional characterization of multiple oligomeric forms of a minimal protein-only ribonuclease P
title_sort bacterial rna-free rnase p: structural and functional characterization of multiple oligomeric forms of a minimal protein-only ribonuclease p
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10652100/
https://www.ncbi.nlm.nih.gov/pubmed/37806495
http://dx.doi.org/10.1016/j.jbc.2023.105327
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