Jerzy Ciesiołka

Identification of the magnesium, europium and lead binding sites in E. coli and lupine tRNAPhe by specific metal ion‐induced cleavages

The Pb, Eu and Mg‐induced cleavages in E. coli and lupine tRNAPhe have been characterized and compared with those found in yeast tRNAPhe. The pattern of lupine tRNAPhe hydrolysis closely resembles that of yeast tRNAPhe, while several major differences occur in the specificity and efficiency of the E. coli tRNAPhe hydrolysis. The latter tRNA is cleaved with much lower yield in the D‐loop, and interestingly, cleavage is also detected in the variable region, that is highly resistant to hydrolysis in eukaryotic tRNAs. The possible location of tight Pb, Eu and Mg binding sites in E. coli tRNAPhe is discussed on the basis of the specific hydrolysis data.

Exploring the minimal substrate requirements for trans-cleavage by RNase P holoenzymes from Escherichia coli and Bacillus subtilis.

We analysed the processing of small bipartite model substrates by Escherichia coli and Bacillus subtilis RNase P and corresponding hybrid enzymes. We demonstrate specific trans‐cleavage of a model substrate with a 4 bp stem and a 1 nucleotide (nt) 5′ flank, representing to date the smallest mimic of a natural RNase P substrate that could be processed in trans at the canonical RNase P cleavage site. Processing efficiencies decreased up to 5000‐fold when the 5′ flank was shortened from 3 to 1 nt. Reduction of the 5′ flank to 1 nt was more deleterious than reducing the stem from 7 to 4 bp, although the 4 bp duplex formed only transiently, in contrast to the stable 7 bp duplex. These results indicate that the crucial contribution of nt −2 in the single‐stranded 5′ flank to productive interaction is a general feature of A‐ and B‐type bacterial RNase P enzymes. We also showed that an Rp‐phosphorothioate modification at nt −2 interferes with processing. Bacterial RNase P holoenzymes are also capable of cleaving single‐stranded RNA oligonucleotides as short as 5 nt, yielding RNase P‐specific 5′‐phosphate and 3′‐OH termini, with measured turnover rates of up to 0.7 min−1. All cleavage sites were at least 2 nt away from the 5′ and 3′ ends of the oligonucleotides. Some cleavage site preferences were observed dependent on the identity of the RNase P RNA subunit.

Structural characterization of the highly conserved 98-base sequence at the 3′ end of HCV RNA genome and the complementary sequence located at the 5′ end of the replicative viral strand

Oligoribonucleotides that corresponded to the X regions of the (+) and (−) polarity strands of HCV RNA, as well as several shorter oligomers comprising defined stem-loop motifs of their predicted secondary structure models, were analyzed by Pb2+-induced cleavage, partial digestion with specific nucleases and chemical modification. Patterns characteristic of the motifs were compared with those obtained for the full-length molecules and on the basis of such ‘structural fingerprinting’ conclusions concerning folding of regions X were formulated. It turned out that the secondary structure model of X(+) RNA proposed earlier, the three-stem-loop model composed of hairpins SL1, SL2 and SL3, was only partially consistent with our experimental data. We confirmed the presence of SL1 and SL3 motifs and showed that the single-stranded stretch adjacent to the earlier proposed hairpin SL2 contributed to the folding of that region. It seemed to be arranged into two hairpins, which might form a hypothetical pseudoknot by changing their base-pairing systems. These data were discussed in terms of their possible biological significance. On the other hand, analysis of the X(−) RNA and its sub-fragments supported a three-stem-loop secondary structure model for this RNA.

Antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method

We have used the in vitro selection method to search for catalytically active variants of the antigenomic delta ribozyme with mutations in the regions that constitute the ribozyme active site: L3, J1/4 and J4/2. In the initial combinatorial library 16 nt positions were randomized and the library contained a full representation of all possible sequences. Following ten cycles of selection-amplification several catalytically active ribozyme variants were identified. It turned out that one-third of the variants contained only single mutation G80U and their activity was similar to that of the wild-type ribozyme. Unexpectedly, in the next one-third of the variants the C76 residue, which was proposed to play a crucial role in the ribozyme cleavage mechanism, was mutated. In these variants, however, a cytosine residue was present in a neighboring position to the polynucleotide chain. It shows that the ribozyme catalytic core possesses substantial ‘structural plasticity’ and the capacity of functional adaptation. Four selected ribozyme variants were subjected to more detailed analysis. It turned out that the variants differed in their relative preferences towards Mg2+, Ca2+ and Mn2+ ions. Thus, the functional properties of the variants were dependent on both the structure of their catalytic sites and divalent metal ions performing catalysis.

Specific RNA cleavages induced by manganese ions.

The specificity and efficiency of manganese ion‐induced RNA hydrolysis was studied with several tRNA molecules. In case of yeast tRNAPhe, the main cleavage occurs at p16 and minor cuts at p17–18, p20–21, p34 and p36–37. The major Mn(II)‐induced cut in yeast elongator tRNAMet is also located in the D‐loop at p16 and it is stronger than that observed in tRNAPhe. In initiator tRNAMet from yeast two strong Mn(II) cleavages of equal intensity occur at p16 and p17. This is in contrast with single, much weaker cuts induced in the D‐loop of that tRNA by Mg(II), Eu(III) and Pb(II) ions. Interestingly, in case of yeast tRNAGlu the main cleavage caused by Mn(II), Mg(II) and Pb(II) ions occurs in the anticodon loop. The involvement of hypermodified base mnm5s2U in this cleavage was ruled out based on results obtained with in vitro transcript of yeast tRNAGlu anticodon arm. Mutation of a single base A37G in the anticodon loop of the transcript drastically reduced the specificity of Mn(II)‐induced hydrolysis.

Interaction of aminoglycosides and their copper(II) complexes with nucleic acids: implication to the toxicity of these drugs

Cupric complexes of eight aminoglycosidic antibiotics were screened for their specific behavior towards tRNAPhe, both in oxidative and neutral surrounding. Without H2O2, the cleavage efficiency was dependent on the resultant charge of the molecule. A comparative assay using tRNAPhe devoid of the natural hypermodification in the anticodon loop proved that hypermodification is indispensable for site recognition and subsequent cleavage. The intensity of single and double strand scissions in plasmid DNA also proceeded in a charge-dependent manner. Unlike free antibiotics, their cupric complexes in the presence of H2O2, facilitated plasmid linearisation and degradation. The participation of ROS in those processes was confirmed using NDMA as a reporter molecule, whose consumption was influenced by the protonation state of the complex.

STRUCTURAL ANALYSIS OF TWO PLANT 5S rRNA SPECIES AND FRAGMENTS THEREOF BY LEAD-INDUCED HYDROLYSIS

The structure of plant 5S rRNA species from lupin and wheat germ as well as the structure of two RNA fragments that represent domains β and γ of lupin 5S rRNA have been probed by Pb(II)‐induced hydrolysis. The lead digestion patterns of 5S rRNA species show that the secondary and tertiary structures of the molecules are very similar. The data suggests that two potential base pairs at the bottom of helix E are destabilized and this causes an enlargement of the hairpin loop e. On the other hand, nucleotides from loop c seem to be involved in the formation of some kind of higher order structure. A comparison of the distribution of cleavages induced in RNA fragments to those in the corresponding regions of the entire 5S rRNA shows that under conditions applied in our studies the structural domains β and γ are not involved in formation of any tertiary interaction within 5S rRNA structure.

Effect of modified nucleotides on structure of yeast tRNAPhe comparative studies by metal ion-induced hydrolysis and nuclease mapping

Structural differences between native yeast tRNA(Phe), its in vitro transcript and the U8G mutant have been investigated using metal ion-induced hydrolysis and nuclease digestion. Differences in the solution structure of the molecules involve four regions: the D- and T-loops, the variable region and the anticodon loop. Efficiency of the Pb(II); Eu(II)-, Mn(II)- and Mg(II)-induced hydrolysis at the main cleavage sites in the D-loop is significantly reduced for unmodified tRNAs. Moreover, only the in vitro transcripts are susceptible for cleavage in the T-loop and entire anticodon loop. Other changes in the transcript molecule involve 50-fold enhancement of S1 nuclease digestion at p36, weak cleavages in the D-loop and lack of some digestion sites in the T-loop. The nuclease V1 digestion patterns are very similar for studied molecules. Changes in the pattern of hydrolysis of the D-loop caused by mutation of the conservative base U8 to G are detected by metal-induced hydrolysis only. Our results indicate clearly that metal ions and enzymatic probes monitor different features of RNA structure and their combined use is highly advantageous in studying subtle structural changes in tRNA.

High affinity of copper(II) towards amoxicillin, apramycin and ristomycin. Effect of these complexes on the catalytic activity of HDV ribozyme.

Three representatives of the distinct antibiotics groups: amoxicillin, apramycin and ristomycin A were studied regarding their impact on hepatitis D virus (HDV) ribozyme both in the metal-free form and complexed with copper(II) ions. Hence the Cu(II)-ristomycin A complex has been characterized by means of NMR, EPR, CD and UV-visible spectroscopic techniques and its binding pattern has been compared with the coordination modes estimated previously for Cu(II)-amoxicillin and Cu(II)-apramycin complexes. It has thus been found that all three antibiotics bind the Cu(II) ion in a very similar manner, engaging two nitrogen and two oxygen donors into coordination with the square planar symmetry in physiological conditions. All three tested antibiotics were able to inhibit the HDV ribozyme catalysis. However, in the presence of the complexes, the catalytic reactions were almost completely inhibited. It was important therefore to check whether the complexes used in lower concentrations could inhibit the HDV ribozyme catalytic activity, thus creating opportunities for their practical application. It turned out that the complexes used in the concentrations of 50μM influenced the catalysis much less effectively comparing to the 200 micromolar concentration. The kobs values were lower than those observed in the control reaction, in the absence of potential inhibitors: 2-fold for amoxicillin, ristomycin A and 3.3-fold for apramycin, respectively.

Phosphate residues of antigenomic HDV ribozyme important for catalysis that are revealed by phosphorothioate modification

The aim of our study was to obtain new details on the role of phosphate residues in antigenomic hepatitis delta virus (HDV) ribozyme for the development of metal ion binding sites and their participation in the cleavage mechanism. In these studies, the wild-type ribozyme and four in vitro selected variants, R37, R20, R25 and R51, were used. The application of nucleotide phosphorothioates and the NAIM (nucleotide analog interference mapping) technique for the cis-acting ribozymes, wild-type, R25 and R51, revealed the importance of the J4/2 and P1.1 regions in the catalysis. Interestingly, in the wild-type ribozyme, the largest interference effects were observed close to catalytic C76 in the presence of Ca2+, while in the case of Mg2+ were in the structurally important helix P1.1. The results obtained for R25 and R51 suggest different coordination of the divalent ions to the phosphate residues within the ribozyme catalytic core. Additionally, replacing the non-bridging oxygen atoms on sulfur in a phosphate group at the cleavage site in trans-acting ribozyme variants showed that interactions between pro-RP and pro-SP oxygen atoms, and catalytic metal ions, had moderate effects on the cleavage reaction. In the wild-type ribozyme, the ratio of SP/RP isomer cleavage rates decreased from 25 for Mg2+-induced cleavage to ca. 4 when thiophilic Mn2+ or Cd2+ were added; thus a “rescue effect” was observed. Interestingly, the R37, R20, R25 and R51 ribozymes showed a reduced RP/SP ratio of cleavage rates and much smaller “rescue effects”. This suggests that the binding of divalent metal ions in the vicinity of the phosphate group at the cleavage site is very sensitive to the overall ribozyme structure.