Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg²⁺ Binding Site is Located Close to the Stacked Branch Adenosine

Abstract

Group II intron self-splicing is essential for correct expression of organellar genes in plants, fungi, and yeast, as well as of bacterial genes. Self-excision of these autocatalytic introns from the primary RNA transcript is achieved in a two-step mechanism apparently analogous to the one of the eukaryotic spliceosome. The 2'-OH of a conserved adenosine (the branch point) located within domain 6 (D6) acts as the nucleophile in the first step of splicing. Despite the biological importance of group II introns, little is known about their structural organization and usage of metal ions in catalysis. Here we report the first solution structure of a catalytically active D6 construct encompassing the branch point and the neighboring helical regions from the mitochondrial yeast intron ai5 . The branch adenosine is the single unpaired nucleotide, and, in contrast to the spliceosomal branch site, resides within the helix being partially stacked between the two flanking GU wobble pairs. We identified a novel prominent Mg2+ binding site in the major groove of the branch site. Importantly, Mg2+ addition does not impair stacking of the branch-adenosine, but in contrast rather strengthens the interaction with the flanking uridines, as shown by NMR-and fluorescence studies. This means, that domain 6 presents the branch adenosine in a stacked fashion to the core of group II introns upon folding to the active conformation.

Metal ion-N7 coordination in a ribozyme branch domain by NMR

“…40,41 The RNA concentration was determined by UV-VIS spectrophotometry, using an extinction coefficient at 260 nm (ε 260 ) of 296. in 3D as the solution structure of D6-27. 29 The branch-adenosine that is stacked within the helix is highlighted in red, the flanking GU-wobble pairs in gold. All previously determined metal ion binding sites are indicated.…”

“…1). 29,30 However for this site, as well as for the whole group II intron architecture, the exact coordination spheres of Mg 2+ ions remain elusive.…”

Exploring Metal Ion Coordination to Nucleic Acids by NMR

“…The latter is used for EPR [38,39] and also NMR studies, where the resonances are severely broadened upon binding of Mn 2+ in the vicinity. [26,40,41] Outer sphere binding of Mg 2+ can be probed with [Co(NH 3 ) 6 ] 3+ (see also Section 3). The ammonia ligands in this kinetically inert complex do not exchange with solvent molecules, allowing them to mimic the [Mg(H 2 O) 6 ] 2+ ion.…”

“…3A). [26,41,57,58] For example, a large chemical shift change is observed for the U19 H1' -A20 H8 crosspeak within domain 6 of a group II intron ribozyme but the line width is unvaried, arguing against a direct coordination of Mg 2+ at the A20 N7 position. [26,60] At the G1 DP , on the other hand, the chemical shift change is accompanied by a significant broadening effect indicating a direct binding of Mg 2+ at the 5'-terminal diphosphate (DP) group.…”

“…[26,60] At the G1 DP , on the other hand, the chemical shift change is accompanied by a significant broadening effect indicating a direct binding of Mg 2+ at the 5'-terminal diphosphate (DP) group. [41] The combination of chemical shift mapping and line broadening studies with Mg 2+ thus yields a rather detailed picture about Mg 2+ binding and accompanying structural changes of the nucleic acid. However, to precisely localize the binding sites as well as the liganding atoms and to determine the geometry of the coordination by NMR, much more information is necessary.…”