Yeast Ssd1 is a non-enzymatic member of the RNase II family with an alternative RNA recognition site.

Ssd1, a conserved fungal RNA-binding protein, is important in stress responses, cell division and virulence. Ssd1 is closely related to Dis3L2 of the RNase II family of nucleases, but lacks catalytic activity and likely suppresses translation of bound mRNAs. Previous studies identified RNA motifs enriched in Ssd1-associated transcripts, yet the sequence requirements for Ssd1 binding are not defined. Here, we identify precise binding sites of Ssd1 on RNA using in vivo cross-linking and cDNA analysis. These sites are enriched in 5' untranslated regions of a subset of mRNAs encoding cell wall proteins. We identified a conserved bipartite motif that binds Ssd1 with high affinity in vitro. Active RNase II enzymes have a characteristic, internal RNA binding path; the Ssd1 crystal structure at 1.9 Å resolution shows that remnants of regulatory sequences block this path. Instead, RNA binding activity has relocated to a conserved patch on the surface of the protein. Structure-guided mutations of this surface prevent Ssd1 from binding RNA in vitro and phenocopy Ssd1 deletion in vivo. These studies provide a new framework for understanding the function of a pleiotropic post-transcriptional regulator of gene expression and give insights into the evolution of regulatory and binding elements in the RNase II family.

Characterization of Highly Efficient RNA-Cleaving DNAzymes that Function at Acidic pH with No Divalent Metal-Ion Cofactors.

Here, we describe the characterization of new RNA-cleaving DNAzymes that showed the highest catalytic efficiency at pH 4.0 to 4.5, and were completely inactive at pH values higher than 5.0. Importantly, these DNAzymes did not require any divalent metal ion cofactors for catalysis. This clearly suggests that protonated nucleic bases are involved in the folding of the DNAzymes into catalytically active structures and/or in the cleavage mechanism. The trans-acting DNAzyme variants were also catalytically active. Mutational analysis revealed a conservative character of the DNAzyme catalytic core that underpins the high structural requirements of the cleavage mechanism. A significant advantage of the described DNAzymes is that they are inactive at pH values close to physiological pH and under a wide range of conditions in the presence of monovalent and divalent metal ions. These pH-dependent DNAzymes could be used as molecular cassettes in biotechnology or nanotechnology, in molecular processes that consist of several steps. The results expand the repertoire of DNAzymes that are active under nonphysiological conditions and shed new light on the possible mechanisms of catalysis.

Impact of Cu(2+) ions on the structure of colistin and cell-free system nucleic acid degradation.

Colistin and transition metal ions are commonly used as feed additives for livestock animals. This work presents the results of an analysis of combined potentiometric and spectroscopic (UV-vis, EPR, CD, NMR) data which lead to conclude that colistin is able to effectively chelate copper(II) ions. In cell-free system the oxidative activity of the complex manifests itself in the plasmid DNA destruction with simultaneous generation of reactive OH species, when accompanied by hydrogen peroxide or ascorbic acid. The degradation of RNA occurs most likely via a hydrolytic mechanism not only for complexed compound but also colistin alone. Therefore, huge amounts of the used antibiotic for nontherapeutic purposes might have a potential influence on livestock health.

In vitro selection of deoxyribozymes active with Cd(2+) ions resulting in variants of DNAzyme 8-17.

In vitro selection was performed to search for RNA-cleaving DNAzymes catalytically active with Cd(2+) ions from the oligonucleotide combinatorial library with a 23-nucleotide random region. All the selected, catalytically active variants turned out to belong to the 8-17 type DNAzyme. Three DNAzymes were prepared in shortened, cis-acting versions which were subjected to a detailed study of the kinetic properties and metal ion preferences. Although the selection protocol was designed for Cd(2+)-dependent DNAzymes, the variants showed broader metal ion specificity. They preferred Cd(2+) but were also active with Mn(2+) and Zn(2+), suggesting that binding of the catalytic ion does not require an extremely specific coordination pattern. The unexpected decrease of the catalytic activity of the variants along with the temperature increase suggested that some changes occurred in their structures or the rate-limiting step of the reaction was changed. Two elements of the catalytic core of DNAzyme 1/VIIWS, the nucleotide at position 12 and the three-base-pair hairpin motif, were mutated. The presence of a purine residue at position 12 was crucial for the catalytic activity but the changes at that position had a relatively small influence on the metal ion preferences of this variant. The middle base pair of the three-base-pair hairpin was changed from A-T to C-G interaction. The catalytic activity of the mutated variant was increased with Zn(2+), decreased with Mn(2+), and was not changed in the presence of Cd(2+) ions. Clearly, this base pair was important for defining the metal ion preferences of the DNAzyme 1/VIIWS.

Antibiotic bacitracin induces hydrolytic degradation of nucleic acids.

BACKGROUND: Bacitracin is a polypeptide antibiotic active against Gram-positive bacterial strains. Its mechanism of action postulates disturbing the cell wall synthesis by inhibiting dephosphorylation of the lipid carrier. We have discovered that bacitracin induces degradation of nucleic acids, being particularly active against RNA. METHODS: In the examination of the nucleolytic activity of bacitracin several model RNA and DNA oligomers were used. The oligomers were labeled at their 5' ends with (32)P radioisotope and following treatment with bacitracin the cleavage sites and efficiency were determined. RESULTS AND CONCLUSIONS: Bacitracin induces degradation of RNA at guanosine residues, preferentially in single-stranded RNA regions. Bacitracin is also able to degrade DNA to some extent but comparable effects to those observed with RNA require its 10-fold higher concentration. The sites of degradation in DNA are very infrequent and preferentially occur near cytidine residues. Free radicals are not involved in the reaction, and which probably proceeds via a hydrolytic mechanism. The phosphate groups at the cleavage sites are present at the 3' ends of RNA products and at the 5' ends of DNA fragments. Importantly, the presence of EDTA does not influence RNA degradation but completely inhibits the degradation of DNA. For DNA degradation divalent metal ions like Mg(2+), Mn(2+) or Zn(2+) are absolutely necessary. GENERAL SIGNIFICANCE: The ability of bacitracin to degrade nucleic acids via a hydrolytic mechanism was a surprising observation, and it is of interest whether these properties can contribute to its mechanisms of action during antibiotic treatment.

Mapping the interactions of selected antibiotics and their Cu2+ complexes with the antigenomic δ ribozyme.

The interactions of selected antibiotics with the trans-acting antigenomic delta ribozyme were mapped. Ribozyme with two oligonucleotide substrates was used, one uncleavable with deoxycytidine at the cleavage site, mimicking the initial state of ribozyme, and the other with an all-RNA substrate mimicking, after cleavage, the product state. Mapping was performed with a set of RNA structural probing methods: Pb(2+) -induced cleavage, nuclease digestion, and the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) approach. The experimental results combined with molecular modeling revealed different binding sites for neomycin B, amikacin and actinomycin D inside the ribozyme structure. Neomycin B, an aminoglycoside antibiotic, which strongly inhibited the catalytic properties of delta ribozyme, was bound to the pocket formed by the P1 stem, the P1.1 pseudoknot, and the J4/2 junction. Amikacin showed less effective binding to the ribozyme catalytic core, resulting in weak inhibition. Complexes of these aminoglycosides with Cu(2+) ions were bound to the same ribozyme regions, but more effectively, showing lower Kd values. On the other hand, the Cu(2+) complex of the cyclopeptide antibiotic actinonomycin D was preferentially intercalated into the P2 and the P4 double-stranded region, and was three times more potent in ribozyme inhibition than the free antibiotic. In addition, some differences in SHAPE reactivities between the ribozyme forms containing all-RNA and deoxycytidine-modified substrates in the J4/2 region were detected, pointing to different ribozyme conformations before and after the cleavage event.

Capreomycin and hygromycin B modulate the catalytic activity of the delta ribozyme in a manner that depends on the protonation and complexation with Cu2+ ions of these antibiotics.

Catalytic RNA molecules (ribozymes) have often been used for the testing of interactions of antibiotics with ribonucleic acids. We showed that the impact of capreomycin and hygromycin B on delta ribozyme catalysis might change dramatically, from stimulation to inhibition, depending on conditions. In order to evaluate possible mechanisms of modulation of the ribozyme catalytic activity we used our earlier data on species distribution for protonated forms of capreomycin and hygromycin B and their complexes with Cu(2+) ions at different pH values. We proposed that, upon inhibition, the protonated amino group of capreomycin was located in the ribozyme catalytic cleft interfering with binding catalytic Mg(2+). Such a mechanism was also supported by the results of ribozyme inhibition with capreomycin complexed with Cu(2+). The effects of stimulation of the delta ribozyme activity by capreomycin and hygromycin B were less pronounced than inhibition. Possibly, the amino functions of these antibiotics might be involved in a general acid-base catalysis performed by the ribozyme, acting as proton acceptors/donors.