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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.

The organometallic complex coenzyme B12 (adenosyl cobalamin, AdoCbl) is not only an essential coenzyme in many biochemical reactions of most if not all living organisms but has lately been shown to play a crucial role in the regulation of B12 related genes. As a consequence, coenzyme B12 has been a target of intense research. However, the investigations of AdoCbl have often been hampered due to its high light-sensitivity leading to decomposition of the compound within a few seconds. Here, we describe a strategy to synthesize more light-stable coenzyme B12 analogs, which show similar steric properties as adenosyl cobalamin. The synthesis, structural characterization as well as the pH dependent “base-on/base-off” behavior of cyanide bridged vitamin B12 conjugates with either a cis-[(NH3)2Pt]2+ or an [enPt]2+ moiety, leading to cis-[(NH3)2PtCl-vitB12]+ (1) and [enPtCl-vitB12]+ (2) are reported. The subsequent reaction of cis-[(NH3)2PtCl-vitB12]+ with the model nucleobase 9-methyladenine leads to the corresponding adduct, where the adenine moiety is coordinated to the Pt2+ center either via N1 or N7. This compound is light-stable and harbors the adenine moiety in the same distance of 5 Å above the corrin plane as present in the highly light-sensitive adenosyl cobalamin.

It is undisputable that the fates of metal ions and nucleic acids are inescapably interwoven. Metal ions are essential for charge compensation of the negatively charged phosphate–sugar backbone, they are instrumental for proper folding, and last but not least they are crucial cofactors for ribozyme catalysis. Considerable progress has been achieved in the past few years on the identification of metal ion binding sites in large DNA and RNA molecules, like in ribozymes including the ribosome. Hereby, most information was gained from crystallography, which fails to explain metal ion binding equilibria in solution as well as the factors that determine the coordination of a metal ion to a specific site. In contrast, such information is readily available for the low-molecular building blocks of large nucleic acids, i.e. for mononucleotides and to some extent also dinucleotides. In this review, we combine and compare for the first time both sets of information. The focus is thereby set on Mg2+, Ca2+, Mn2+, and Cd2+ because these four metal ions are either freely available in cells, have a large impact on the catalytic rate of ribozymes, and/or are often applied in RNA biochemistry. Our comparisons show that results obtained from small molecules can be directly transposed to the findings in large RNA structures like the ribosome. For example, the basic coordination-chemical properties of the different metal ions are reflected in their binding to large nucleic acid structures: macrochelate formation, e.g. the simultaneous intranucleotide coordination of a Mg2+ ion to the phosphate unit and the N7 site of a purine nucleobase (be it inner- or outersphere), is well known for mononucleotides. We show that the frequency of occurrence of this type of coordination is the same for mononucleotides and the ribosome.

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A series of pyrazine (pz) complexes containing cis-(NH(3))(2)Pt(II), (tmeda)Pt(II) (tmeda = N,N,N',N'-tetramethylethylenediamine), and trans-(NH(3))(2)Pt(II) entities have been prepared and characterized by X-ray crystallography and/or (1)H NMR spectroscopy. In these compounds, the pz ligands act as monodentate (1-3) or bidentate bridging ligands (4-7). Three variants of the latter case are described: a dinuclear complex [Pt(II)](2) (4b), a cyclic tetranuclear [Pt(II)](4) complex (5), and a trinuclear mixed-metal complex [Pt(2)Ag] (7). Mono- and bidentate binding modes are readily differentiated by (1)H NMR spectroscopy, and the assignment of pz protons in the case of monodentate coordination is aided by the observation of (195)Pt satellites. Formation of the open molecular box cis-[{(NH(3))(2)Pt(pz)}(4)](NO(3))(8).3.67H(2)O (5) from cis-(NH(3))(2)Pt(II) and pz follows expectations of the "molecular library approach" for the generation of a cyclic tetramer.

Naturally occurring RNA molecules exhibit many unexpected and fascinating properties in living cells like protein synthesis and transport, regulation of metabolic functions, and catalytic cleavage reactions. To understand this functional diversity, a detailed knowledge of RNA structure and metal ion-binding properties is crucial. In our research group, we address these problems by combining various biochemical, analytical and spectroscopic techniques. A large part of our work is devoted to the structure determination of catalytic RNA molecules, i.e. ribozymes, by NMR. Based on the three-dimensional structure, further experiments are carried out to understand in detail the effects of different metal ions on the local and global structure, as well as catalysis itself.

In vitro run-off transcription from a double-stranded DNA template by T7 RNA polymerase is an elegant way to obtain highly pure and uniform RNA oligonucleotides of lengths ranging from about 15 to several thousand nucleotides. Here we describe the different strategies applied and optimized in our laboratory to enzymatically synthesize RNAs as necessary when working at the interface of bioinorganic chemistry, coordination chemistry, RNA biochemistry and structural biology.

Domain 5 (D5) is the central core of group II intron ribozymes. Many base and backbone substituents of this highly conserved hairpin participate in catalysis and are crucial for binding to other intron domains. We report the solution structures of the 34-nucleotide D5 hairpin from the group II intron ai5 gamma in the absence and presence of divalent metal ions. The bulge region of D5 adopts a novel fold, where G26 adopts a syn conformation and flips down into the major groove of helix 1, close to the major groove face of the catalytic AGC triad. The backbone near G26 is kinked, exposing the base plane of the adjacent A-U pair to the solvent and causing bases of the bulge to stack intercalatively. Metal ion titrations reveal strong Mg(2+) binding to a minor groove shelf in the D5 bulge. Another distinct metal ion-binding site is observed along the minor groove side of the catalytic triad, in a manner consistent with metal ion binding in the ribozyme active site.

Methylation at the N7 position is one of the most frequently naturally occurring modifications of guanosine. This alteration drastically changes the hydrogen bonding and acid–base properties of the guanine nucleobase. Here we show on the example of the model nucleobase 7,9-dimethylguanine that due to blockage of N7 of the purine ring, new hydrogen bonding patterns occur on the minor groove binding face of this nucleobase involving the ring nitrogen N3 and the exocyclic amino group N2H2. The free 7,9-dimethylguaninium ion and several transplatinum(II) complexes of the this ligand are presented and discussed.