Divalent Metal Ions Tune the Self-Splicing Reaction of the Yeast Mitochondrial Group II Intron Sc.ai5γ

Group II introns are large ribozymes, consisting of six functionally distinct domains that assemble in the presence of Mg2+ to the active structure catalyzing a variety of reactions. The first step of intron splicing is well characterized by a Michaelis–Menten-type cleavage reaction using a two-piece group II intron: the substrate RNA, the 5′-exon covalently linked to domains 1, 2, and 3, is cleaved upon addition of domain 5 acting as a catalyst. Here we investigate the effect of Ca2+, Mn2+, Ni2+, Zn2+, Cd2+, Pb2+, and [Co(NH3)6]3+ on the first step of splicing of the Saccharomyces cerevisiae mitochondrial group II intron Sc.ai5γ. We find that this group II intron is very sensitive to the presence of divalent metal ions other than Mg2+. For example, the presence of only 5% Ca2+ relative to Mg2+ results in a decrease in the maximal turnover rate k cat by 50%. Ca2+ thereby has a twofold effect: this metal ion interferes initially with folding, but then also competes directly with Mg2+ in the folded state, the latter being indicative of at least one specific Ca2+ binding pocket interfering directly with catalysis. Similar results are obtained with Mn2+, Cd2+, and [Co(NH3)6]3+. Ni2+ is a much more powerful inhibitor and the presence of either Zn2+ or Pb2+ leads to rapid degradation of the RNA. These results show a surprising sensitivity of such a large multidomain RNA on trace amounts of cations other than Mg2+ and raises the question of biological relevance at least in the case of Ca2+

Optimal molecular crowding accelerates group II intron folding and maximizes catalysis

Unlike in vivo conditions, group II intron ribozymes are known to require high magnesium(II) concentrations ([Mg2+]) and high temperatures (42 °C) for folding and catalysis in vitro. A possible explanation for this difference is the highly crowded cellular environment, which can be mimicked in vitro by macromolecular crowding agents. Here, we combined bulk activity assays and single-molecule Förster Resonance Energy Transfer (smFRET) to study the influence of polyethylene glycol (PEG) on catalysis and folding of the ribozyme. Our activity studies reveal that PEG reduces the [Mg2+] required, and we found an “optimum” [PEG] that yields maximum activity. smFRET experiments show that the most compact state population, the putative active state, increases with increasing [PEG]. Dynamic transitions between folded states also increase. Therefore, this study shows that optimal molecular crowding concentrations help the ribozyme not only to reach the native fold but also to increase its in vitro activity to approach that in physiological conditions

Influence of decreasing solvent polarity (1,4-dioxane/water mixtures) on the stability and structure of complexes formed by copper(II), 2,2`-bipyridine or 1,10-phenanthroline and guanosine 5`-diphosphate : evaluation of isomeric equilibria

The stability constants of the 1 : 1 complexes formed between Cu(Arm)2+, where Arm = 2,2`-bipyridine or 1,10-phenanthroline, and guanosine 5`-diphosphate (GDP)3- or its monoprotonated form H(GDP)2- were determined by potentiometric pH titrations in water and in water containing 30 or 50 stability of the binary Cu(GDP)- complex is enhanced due to macrochelate formation of the diphosphate-coordinated Cu2+ with N7 of the guanine residue as previously shown. In Cu(Arm)(GDP)- the N7 is released from Cu2+ and the stability enhancement of more than one log unit in aqueous solution is clearly attributable to intramolecular stack formation between the aromatic rings of Arm and the guanine moiety. Indeed, stacked isomers occur to more than 90 with open unstacked forms. Surprisingly, the same formation degrees of the stacks are observed for Cu(Arm)(dGMP) complexes, where dGMP2- = 2`-deoxyguanosine 5`-monophosphate, despite the fact that the overall stability of the latter species is by about 2.7 log units lower. In 1,4-dioxane-water mixtures stack formation is drastically reduced, probably due to hydrophobic solvation of the aromatic rings by the ethylene bridges of 1,4-dioxane. The relevance of these results regarding biological systems is indicated

Acid-base and metal ion binding properties of 2-thiocytidine in aqueous solution

The thionucleoside 2-thiocytidine (C2S) occurs in nature in transfer RNAs; it receives attention in diverse fields like drug research and nanotechnology. By potentiometric pH titrations we measured the acidity constants of H(C2S)(+) and the stability constants of the M(C2S)(2+) and M(C2S-H)(+) complexes (M2+ = Zn2+ , Cd2+), and we compared these results with those obtained previously for its parent nucleoside, cytidine (Cyd). Replacement of the (C2)=O unit by (C2)=S facilitates the release of the proton from (N3)H+ in H(C2S)(+) (pK (a) = 3.44) somewhat, compared with H(Cyd)(+) (pK (a) = 4.24). This moderate effect of about 0.8 pK units contrasts with the strong acidification of about 4 pK units of the (C4)NH2 group in C2S (pK (a) = 12.65) compared with Cyd (pK (a) approximate to 16.7); the reason for this result is that the amino-thione tautomer, which dominates for the neutral C2S molecule, is transformed upon deprotonation into the imino-thioate form with the negative charge largely located on the sulfur. In the M(C2S)(2+) complexes the (C2)S group is the primary binding site rather than N3 as is the case in the M(Cyd)(2+) complexes, though owing to chelate formation N3 is to some extent still involved in metal ion binding. Similarly, in the Zn(C2S-H)(+) and Cd(C2S-H)(+) complexes the main metal ion binding site is the (C2)S- unit (formation degree above 99.99 chelate formation with N3 must be surmised for the M(C2S-H)(+) species in accord with previous solid-state studies of related ligands. Upon metal ion binding, the deprotonation of the (C4)NH2 group (pK(a) = 12.65) is dramatically acidified (pK (a) approximate to 3), confirming the very high stability of the M(C2S-H)(+) complexes. To conclude, the hydrogen-bonding and metal ion complex forming capabilities of C2S differ strongly from those of its parent Cyd; this must have consequences for the properties of those RNAs which contain this thionucleoside

Effects of N7-methylation, N7-platination, and C8-hydroxylation of guanine on H-bond formation with cytosine: platinum coordination strengthens the Watson-Crick pair.

The hydrogen bonding properties of 1-methylcytosine (1-MeC) with the following guanine base derivatives have been studied in DMSO-d6, applying concentration-dependent 1H NMR spectroscopy: 9-ethylguanine, 7,9-dimethylguanine (7,9-DimeGH+), and 7,8-dihydro-8-oxo-9-methylguanine (8-O-9-MeGH), as well as three 9-ethylguanine complexes carrying different Pt(II) moieties at the N7 position. The association constants K for the Watson-Crick pairing schemes are by a factor 2-3 higher in the cases of platinated guanine complexes compared to the Watson-Crick pair between 9-ethylguanine and 1-methylcytosine (K = 6.9 +/- 1.3 M(-1)). Similar enhanced stabilities are observed for the pairs formed between 1-MeC and 7,9-DimeGH+ or 8-O-9-MeGH. The increase in N1H acidity of the guanine derivative upon modification at the N7 or C8 positions can be correlated with the association constants K; the result is a bell-shaped curve meaning that acidification initially stabilizes hydrogen bond formation up to a certain maximum; further acidification then leads to a destabilization. For two of the examples studied in solution, hydrogen bonding according to Watson-Crick between N7-platinated 9-ethylguanine and 1-methylcytosine has also been established by X-ray crystallography