Abstract
All species transcribe ribosomal RNA in an immature form that requires several enzymes for processing into mature rRNA. The number and types of enzymes utilized for these processes vary greatly between different species. In low G + C Gram-positive bacteria including Bacillus subtilis and Geobacillus stearothermophilus, the endoribonuclease (RNase) M5 performs the final step in 5S rRNA maturation, by removing the 3'- and 5'-extensions from precursor (pre) 5S rRNA. This cleavage activity requires initial complex formation between the pre-rRNA and a ribosomal protein, uL18, making the full M5 substrate a ribonucleoprotein particle (RNP). M5 contains a catalytic N-terminal Toprim domain and an RNA-binding C-terminal domain, respectively, shown to assist in processing and binding of the RNP. Here, we present structural data that show how two Mg2+ ions are accommodated in the active site pocket of the catalytic Toprim domain and investigate the importance of these ions for catalysis. We further perform solution studies that support the previously proposed 3'-before-5' order of removal of the pre-5S rRNA extensions and map the corresponding M5 structural rearrangements during catalysis.
Keywords:
5S; M5; Ribosomal RNA; Toprim domain; maturation; mechanism; processing; ribonuclease.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Bacillus subtilis / enzymology*
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Endoribonucleases / chemistry*
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Endoribonucleases / genetics
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Endoribonucleases / metabolism*
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Geobacillus stearothermophilus / enzymology*
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Magnesium / metabolism*
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Nucleic Acid Conformation
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RNA Precursors / genetics
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RNA Precursors / metabolism*
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RNA, Double-Stranded / genetics
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RNA, Double-Stranded / metabolism*
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RNA, Ribosomal, 5S / genetics
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RNA, Ribosomal, 5S / metabolism*
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Ribosomes / genetics
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Ribosomes / metabolism
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Substrate Specificity
Substances
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RNA Precursors
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RNA, Double-Stranded
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RNA, Ribosomal, 5S
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Endoribonucleases
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ribonuclease M5
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Magnesium
Grants and funding
This work was supported by the Centre National de la Recherche Scientifique (CNRS, UMR8261), the Agence Nationale de la Recherche (Project ARNr-QC, ANR-15-CE11-0009) and the Labex DYNAMO (ANR-11-LABX- 0011). SC and MB thank the GIS IBISA and the Région Alsace for financial support for Synapt G2 HDMS instrumental purchase, and MB thanks the Région Alsace for financial support. The authors acknowledge the support and the use of resources of the French Proteomic Infrastructure ProFI ANR-10-INBS-08-03. The authors thank the University of Strasbourg (IdeX Program), the GIS IBiSA and Région Alsace for financial support in purchasing a Synapt G2SI HDMS instrument. MB was supported by a fellowship from the Région Alsace.