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Specific phosphorothioate substitution within domain 6 of a group II intron ribozyme leads to changes in local structure and metal ion binding

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Author Erat Michèle C., Besic Emina, Oberhuber Michael, Johannsen Silke, Sigel Roland K. O.,
Project Metal Ions in Structure and Function of Regulatory RNAs
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Original article (peer-reviewed)

Journal JBIC Journal of Biological Inorganic Chemistry
Volume (Issue) 23(1)
Page(s) 167 - 177
Title of proceedings JBIC Journal of Biological Inorganic Chemistry
DOI 10.1007/s00775-017-1519-3

Abstract

Group II introns are large self-splicing ribozymes that require high amounts of monovalent and divalent metal ions for folding and catalysis under in vitro conditions. Domain 6 of these ribozymes contains a highly conserved adenosine whose 2′-OH acts as a nucleophile during self-cleavage via the branching pathway. We have previously suggested a divalent metal ion that binds to the major groove at the GU wobble pair above the branch-A in a minimal, but active branch domain construct (D6–27) from the yeast mitochondrial intron Sc.ai5γ. Here we characterize metal ion binding to the phosphate oxygens at the branch site. In vitro transcription yielded a D6–27 construct where all R P oxygens of the uridine phosphate groups are replaced by sulfur (α-thio-D6–27). We determined its NMR structure, the second RNA-only structure containing thiophosphate groups. [31P] resonances were assigned and chemical shift changes monitored upon titration with Cd2+. In addition, the two uridines flanking the branch-point, U19 and U21 were specifically thioated by chemical synthesis (thio-U19-D6–27 and thio-U19/U21-D6–27), enabling us to study Cd2+ binding at the R P-, as well as the S P- position of the corresponding phosphate oxygens. Our studies reveal that both non-bridging phosphate oxygens of U19 are involved in metal ion coordination, whereas only the major groove phosphate oxygen of U21 is influenced. Together with NOE data of a hexaamminecobalt(III) titration, this suggests a single metal ion binding site at the GU wobble pair above the branch point in the major groove of D6 of this group II intron ribozyme.
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