Role of divalent metal ions in the interaction of RNA with small molecules

We aim to understand the role divalent metal ions play in the binding of RNA to small ligands. This problem will be addressed in four approaches: i) An aminoglycoside antibiotic-binding site in a group I intron will be functionally compared to its equivalent binding site in the ribosome. We have postulated several years ago, that splice site selection in group I introns is functionally related to the decoding process at the ribosome . The 16S rRNA decoding site, also called A-site, has been dissected and inserted into the group I intron core replacing the functionally equivalent P4/P5 domain. This resulted in a functional intron. Inhibition of splicing by the aminoglycoside neomycm B is most probably due to displacement of a magnesium ion which lies at the interface between the catalytic core of the intron and the P4/P5 domain. The role of this metal ion in splicing and splicing inhibition will be studied in detail. ii) The P7 stem located in the catalytic core of self-splicing introns contains an unpaired base (bulged nucleotide), which is semi-conserved. A or C at this position supports active catalysis. We have proposed that this base (the N3 position of C or the N1 position of A) co-ordinates a metal ion, responsible for catalysis. We now wish to introduce, 2-aminopurine, into this position. Metal ion dependent folding, and/or ligand binding (guanosine cofactor, substrate, antibiotics) will be monitored by changes in the fluorescence intensity, anisotropy, or lifetime of the site-specific fluorescent tracer within the 265 nt long RNA. These experiments will allow a detailed approach in the analyses of structure and dynamics of folding and catalysis of the td ribozyme. iii) Self-cleavage of the hammerhead and the human Hepatitis Delta Virus ribozymes is inhibited by several antibiotics. Inhibition of catalysis appears to be directly correlated with divalent metals ions, which are probably displaced by the antibiotics. An extensive kinetic analysis using a wide range of divalent metal ions will be undertaken to elucidate the mechanism of action of tetracycline in inhibiting catalysis of small ribozymes. iv) We have isolated via in vitro selection small RNA aptamers which specifically bind streptomycin. One of these RNAs is especially interesting because it discriminates by at least four orders of magnitude between streptomycin and a structurally related compound, bluensomycin. The binding of streptomycin to this RNA requires Mg 2+. We intend to analyse the kinetic and thermodynamic properties of this co-operative and discriminative binding.

Ribonucleic acid (RNA) molecules are able to fold into complex three-dimensional structures, which form binding sites for small ligands like antibiotics. Divalent metal ions play a key role in the formation of three-dimensional structures and in ligand binding. In the current project we studied the interaction of small molecules with RNA focussing on the role of Mg2+ in the binding process. Several approaches were undertaken to study this fundamental property of RNA molecules. Since large RNA molecules are composed of multiple independent domains, we dissected the decoding site of the 16S ribosomal RNA, the most prominent antibiotic-binding site of the ribosome, and swapped it into a group I intron. This experiment revealed the substrate binding characteristics of that special group I intron. Substrate binding is inhibited by a "folding problem" due to the alternative base pairing propensities of two hairpins, P1 and P2. The two hairpins are in competition, leading to a rate limiting folding event in the activity of this RNA. We further focussed on the evaluation of a new method, which is useful for monitoring binding of divalent ions to RNA molecules. This method consisted in using uranyl during folding and irradiating the sample with a 420 nm wavelength light, which leads to backbone cleavage in the vicinity of the uranyl ion. This method was analysed with five RNAs, whose crystal structures are known, enabling a good evaluation of the method. We concluded that the method is only partially valid, since not all magnesium binding sites are replaced by uranyl, but that the method is very good for detecting flexible regions in a large molecule. A mutant intron, which affects binding of a potentially catalytic metal ion was further analysed with a kinetic approach. This enabled the detection of two phenotypes, rescue of the phenotype with mangenese or with elevation of the pH. This suggests, that the mutated position is involved in the activation of the guanosine cofactor for nucleophillic attack. Our last approach consisted in using spectroscopy to monitor binding of antibiotics to RNA molecules. Small in vitro selected antibiotic binding RNAs were used in UV-melting and CD spectra as well as in fluorescent spectroscopy. These experiments are a promising start for future projects and could give a first insight into the potential of spectroscopic methods for RNA-ligand chemistry.