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  • 1
    Publikationsdatum: 2013-10-15
    Beschreibung: Post-transcriptional modifications are essential to the cell life cycle, as they affect both pre-ribosomal RNA processing and ribosome assembly. The box C/D ribonucleoprotein enzyme that methylates ribosomal RNA at the 2'-O-ribose uses a multitude of guide RNAs as templates for the recognition of rRNA target sites. Two methylation guide sequences are combined on each guide RNA, the significance of which has remained unclear. Here we use a powerful combination of NMR spectroscopy and small-angle neutron scattering to solve the structure of the 390 kDa archaeal RNP enzyme bound to substrate RNA. We show that the two methylation guide sequences are located in different environments in the complex and that the methylation of physiological substrates targeted by the same guide RNA occurs sequentially. This structure provides a means for differential control of methylation levels at the two sites and at the same time offers an unexpected regulatory mechanism for rRNA folding.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Lapinaite, Audrone -- Simon, Bernd -- Skjaerven, Lars -- Rakwalska-Bange, Magdalena -- Gabel, Frank -- Carlomagno, Teresa -- England -- Nature. 2013 Oct 24;502(7472):519-23. doi: 10.1038/nature12581. Epub 2013 Oct 13.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/24121435" target="_blank"〉PubMed〈/a〉
    Schlagwort(e): Apoproteins/chemistry/metabolism ; Archaeal Proteins/chemistry/metabolism ; Biocatalysis ; Chromosomal Proteins, Non-Histone/metabolism ; Methylation ; Models, Molecular ; Multiprotein Complexes/chemistry/metabolism ; Nucleic Acid Conformation ; Pyrococcus furiosus/*enzymology/*genetics ; RNA Folding ; *RNA Processing, Post-Transcriptional ; RNA, Archaeal/chemistry/genetics/metabolism ; RNA, Guide/chemistry/genetics/metabolism ; RNA, Ribosomal/*chemistry/*metabolism ; Ribonucleoproteins, Small Nucleolar/*chemistry/*metabolism
    Print ISSN: 0028-0836
    Digitale ISSN: 1476-4687
    Thema: Biologie , Chemie und Pharmazie , Medizin , Allgemeine Naturwissenschaft , Physik
    Standort Signatur Erwartet Verfügbarkeit
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  • 2
    Publikationsdatum: 2020-02-12
    Beschreibung: Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners.
    Materialart: info:eu-repo/semantics/article
    Format: application/pdf
    Standort Signatur Erwartet Verfügbarkeit
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