6EZ0

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

Summary for 6EZ0

• Entry DOI: 10.2210/pdb6ez0/pdb
• Related: 2AHT
• NMR Information: BMRB: 34199
• Descriptor: RNA (27-MER) (1 entity in total)
• Functional Keywords: thiophosphate group ii intron branch-adenosine hairpin tetraloop, rna
• Biological source: Saccharomyces cerevisiae
• Total number of polymer chains: 1
• Total formula weight: 8800.56

Authors

Erat, M.C.,Besic, E.,Oberhuber, M.,Johannsen, S.,Sigel, R.K.O. (deposition date: 2017-11-13, release date: 2018-01-03, Last modification date: 2024-06-19)

Primary citation

Erat, M.C.,Besic, E.,Oberhuber, M.,Johannsen, S.,Sigel, R.K.O.
Specific phosphorothioate substitution within domain 6 of a group II intron ribozyme leads to changes in local structure and metal ion binding.
J. Biol. Inorg. Chem., 23:167-177, 2018

PubMed 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 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. [P] resonances were assigned and chemical shift changes monitored upon titration with Cd. 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 Cd binding at the R -, as well as the S - 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.

PubMed: 29218637
DOI: 10.1007/s00775-017-1519-3

Experimental method

SOLUTION NMR