Bernd Knobloch

The structural stabilization of the κ three-way junction by Mg(II) represents the first step in the folding of a group II intron

Folding of group II introns is characterized by a first slow compaction of domain 1 (D1) followed by the rapid docking of other domains to this scaffold. D1 compaction initiates in a small subregion encompassing the κ and ζ elements. These two tertiary elements are also the major interaction sites with domain 5 to form the catalytic core. Here, we provide the first characterization of the structure adopted at an early folding step and show that the folding control element can be narrowed down to the three-way junction with the κ motif. In our nuclear magnetic resonance studies of this substructure derived from the yeast mitochondrial group II intron Sc.ai5γ, we show that a high affinity Mg(II) ion stabilizes the κ element and enables coaxial stacking between helices d′ and d′′, favoring a rigid duplex across the three-way junction. The κ-element folds into a stable GAAA-tetraloop motif and engages in A-minor interactions with helix d′. The addition of cobalt(III)hexammine reveals three distinct binding sites. The Mg(II)-promoted structural rearrangement and rigidification of the D1 core can be identified as the first micro-step of D1 folding.

Discrimination in metal-ion binding to RNA dinucleotides with a non-bridging oxygen or sulfur in the phosphate diester link.

Replacement of a non-bridging oxygen in the phosphate diester bond by a sulfur has become quite popular in nucleic acid research and is often used as a probe, for example, in ribozymes, where the normally essential Mg(2+) is partly replaced by a thiophilic metal ion to reactivate the system. Despite these widely applied rescue experiments no detailed studies exist quantifying the affinity of metal ions to such terminal sulfur atoms. Therefore, we performed potentiometric pH titrations to determine the binding properties of pUp((S))U(3-) towards Mg(2+), Mn(2+), Zn(2+), Cd(2+), and Pb(2+), and compared these data with those previously obtained for the corresponding pUpU(3-) complexes. The primary binding site in both dinucleotides is the terminal phosphate group. Theoretically, also the formation of 10-membered chelates involving the terminal oxygen or sulfur atoms of the (thio)phosphate bridge is possible with both ligands. The results show that Mg(2+) and Mn(2+) exist as open (op) isomers binding to both dinucleotides only at the terminal phosphate group. Whereas Cd(pUpU)(-) only exists as Cd(pUpU)(-)(op), Cd(pUp((S))U)(-) is present to about 64 % as the S-coordinated macrochelate, Cd(pUp((S))U)(-)(cl/PS). Zn(2+) forms with pUp((S))U(3-) three isomeric species, that is, Zn(pUp((S))U)(-)(op), Zn(pUp((S))U)(-)(cl/PO), and Zn(pUp((S))U)(-)(cl/PS), which occur to about 33, 12 (O-bound), and 55 %, respectively. Pb(2+) forms the 10-membered chelate with both nucleotides involving only the terminal oxygen atoms of the (thio)phosphate bridge, that is, no indication of S binding was discovered in this case. Hence, Zn(2+) and Cd(2+) show pronounced thiophilic properties, whereas Mg(2+), Mn(2+), and Pb(2+) coordinate to the oxygen, macrochelate formation being of relevance with Pb(2+) only.