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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.
Affiliation
  • Wilhelm CA; Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
  • Mallik L; Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA; Center for Computational and Genomic Medicine and Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
  • Kelly AL; Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
  • Brotzman S; Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
  • Mendoza J; Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
  • Anders AG; Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
  • Leskaj S; Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
  • Castillo C; Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
  • Ruotolo BT; Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
  • Cianfrocco MA; Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA; Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, USA.
  • Koutmos M; Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA; Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA; Program in Biophysics, University of Michigan, Ann Arbor, Michigan, USA. Electronic address: mkoutmos@umich.edu.
J Biol Chem ; 299(11): 105327, 2023 Nov.
Article in En | MEDLINE | ID: mdl-37806495
ABSTRACT
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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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: RNA, Bacterial / Ribonuclease P Language: En Journal: J Biol Chem Year: 2023 Document type: Article Affiliation country: United States

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: RNA, Bacterial / Ribonuclease P Language: En Journal: J Biol Chem Year: 2023 Document type: Article Affiliation country: United States