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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


Erat, Michèle C; Besic, Emina; Oberhuber, Michael; Johannsen, Silke; Sigel, Roland K O (2018). Specific phosphorothioate substitution within domain 6 of a group II intron ribozyme leads to changes in local structure and metal ion binding. Journal of Biological Inorganic Chemistry, 23(1):167-177.

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.

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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Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Language:English
Date:2018
Deposited On:08 Feb 2018 11:23
Last Modified:20 Feb 2018 09:05
Publisher:Springer
ISSN:0949-8257
OA Status:Closed
Publisher DOI:https://doi.org/10.1007/s00775-017-1519-3

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