Studies of viomycin, an anti-tuberculosis antibiotic: copper(ii) coordination, DNA degradation and the impact on delta ribozyme cleavage activity.

Viomycin is a basic peptide antibiotic, which is among the most effective agents against multidrug-resistant tuberculosis. In this paper we provide the characteristics of its acid base properties, coordination preferences towards the Cu(ii) ions, as well as the reactivity of the resulting complexes against plasmid DNA and HDV ribozyme. Careful coordination studies throughout the wide pH range allow for the characterisation of all the Cu(ii)-viomycin complex species. The assignment of proton chemical shifts was achieved by NMR experiments, while the DTF level of theory was applied to support molecular structures of the studied complexes. The experiments with the plasmid DNA reveal that at the physiological levels of hydrogen peroxide the Cu(ii)-viomycin complex is more aggressive against DNA than uncomplexed metal ions. Moreover, the degradation of DNA by viomycin can be carried out without the presence of transition metal ions. In the studies of antigenomic delta ribozyme catalytic activity, viomycin and its complex are shown to modulate the ribozyme functioning. The molecular modelling approach allows the indication of two different locations of viomycin binding sites to the ribozyme.

Lifetime measurements of the neutron-rich N = 30 isotones 50Ca and 51Sc: orbital dependence of effective charges in the fp shell.

The lifetimes of the first excited states of the N = 30 isotones (50)Ca and (51)Sc have been determined using the Recoil Distance Doppler Shift method in combination with the CLARA-PRISMA spectrometers. This is the first time such a method is applied to measure lifetimes of neutron-rich nuclei populated via a multinucleon transfer reaction. This extends the lifetime knowledge beyond the f_{7/2} shell closure and allows us to derive the effective proton and neutron charges in the fp shell near the doubly magic nucleus (48)Ca, using large-scale, shell-model calculations. These results indicate an orbital dependence of the core polarization along the fp shell.

Effective charge of the (pi)h(11/2) orbital and the electric field gradient of Hg from the Yrast structure of 206Hg.

The gamma-ray decay of excited states of the two-proton hole nucleus, 206Hg, has been identified using Gammasphere and 208Pb+238U collisions. The yrast states found include a T(1/2) = 92(8) ns 10(+) isomer located above the known 5(-) isomer. The B(E2;10(+)-->8(+)) strength is used to derive the quadrupole polarization charge induced by the h(11/2) proton hole. Also, the implied quadrupole moment has been used to provide an absolute scale for the electric field gradient of Hg in Hg metal.

Ribozymes of the hepatitis delta virus: recent findings on their structure, mechanism of catalysis and possible applications.

Although the delta ribozymes have been studied for more than ten years the most important information concerning their structure and mechanism of catalysis were only obtained very recently. The crystal structure of the genomic delta ribozyme turns out to be an excellent example of the extraordinary properties of RNA molecules to fold into uniquely compact structures. Details of the X-ray structure have greatly stimulated further studies on the folding of the ribozymes into functionally active molecules as well as on the mechanism of RNA catalysis. The ability of the delta ribozymes to carry out general acid-base catalysis by nucleotide side chains has been assumed in two proposed mechanisms of self-cleavage. Recently, considerable progress has been also made in characterizing the catalytic properties of trans-acting ribozyme variants that are potentially attractive tools in the strategy of directed RNA degradation.

Catalytic cleavage of cis- and trans-acting antigenomic delta ribozymes in the presence of various divalent metal ions.

Catalytic activity of four structural variants of the antigenomic delta ribozyme, two cis- and two trans-acting, has been compared in the presence of selected divalent metal ions that effectively support catalysis. The ribozymes differ in regions that are not directly involved in formation of the ribozyme active site: the region immediately preceding the catalytic cleavage site, the P4 stem and a stretch of the viral RNA sequence extending the minimal ribozyme sequence at its 3'-terminus. The variants show high cleavage activity in the presence of Mg(2+), Ca(2+) and Mn(2+), lower with Co(2+) and Sr(2+) and some variants are also active with Cd(2+) and Zn(2+) ions. In the presence of a particular metal ion the ribozymes cleave, however with different initial rates, according to pseudo-first or higher order kinetics and to different final cleavage extents. On the other hand, relatively small differences are observed in the reactions induced by various metal ions. The cleavage of trans-acting ribozymes induced by Mg(2+) is partially inhibited in the presence of Na(+), spermidine and some other divalent metal ions. The inert Co(NH(3))(6)(3+) complex is unable to support catalysis, as reported earlier for the genomic ribozyme. The results are discussed in terms of the influence of structural elements peripheral to the ribozyme active site on its cleavage rate and efficiency as well as the role of metal ions in the cleavage mechanism. Some implications concerning further studies and possible applications of delta ribozymes are also considered.

Isolation and properties of Escherichia coli 23S-RNA pseudouridine 1911, 1915, 1917 synthase (RluD).

All nine pseudouridine (psi) residues in Escherichia coli 23S RNA are in or very near the peptidyl transfer centre (PTC) of the ribosome. Five psi synthases catalyze synthesis of these nine psi's. Deletion of the gene for one psi synthase, RluD, which directs synthesis of three closely clustered psi's in the decoding site of the PTC, has a profound negative impact on cell growth. We describe the isolation, without amplification from a cloned coding element, of the triple-site modifying enzyme, RluD, the N-terminal sequence of which has been used to clone and express the corresponding gene, rluD. Unlike "expressed" RluD, which so far has not been shown to modify one (1911) of the three closely clustered sites (1911, 1915, 1917), "natural" RluD modifies all three sites; and unlike another pai synthase, RluA, natural RluD has greatly expanded modifying activity at low Mg concentrations. These properties of the expressed and natural forms of RluD are discussed.

Mapping of accessible sites for oligonucleotide hybridization on hepatitis delta virus ribozymes.

Semi-random libraries of DNA 6mers and RNase H digestion were applied to search for sites accessible to hybridization on the genomic and antigenomic HDV ribozymes and their 3' truncated derivatives. An approach was proposed to correlate the cleavage sites and most likely sequences of oligomers, members of the oligonucleotide libraries, which were engaged in the formation of RNA-DNA hybrids. The predicted positions of oligomers hybridizing to the genomic ribozyme were compared with the fold of polynucleotide chain in the ribozyme crystal structure. The data exemplified the crucial role of target RNA structural features in the binding of antisense oligonucleotides. It turned out that cleavages were induced if the bound oligomer could adapt an ordered helical conformation even when it required partial penetration of an adjacent double-stranded region. The major features of RNA structure disfavoring hybridization and/or RNase H hydrolysis were sharp turns of the polynucleotide chain and breaks in stacking interactions of bases. Based on the predicted positions of oligomers hybridizing to the antigenomic ribozyme we chose and synthesized four antisense DNA 6mers which were shown to direct hydrolysis in the desired, earlier predicted regions of the molecule.

Sequential folding of the genomic ribozyme of the hepatitis delta virus: structural analysis of RNA transcription intermediates.

The structures of the model oligoribonucleotides that mimic the consecutive stages in the transcription of genomic HDV ribozyme have been analyzed by the Pb(2+)-induced cleavage method, partial digestion with specific nucleases and chemical probing. In the transcription intermediates, the P1 and P4 helical segments are found to be present in the final folded forms in which they exist in the full-length transcript. However, the region corresponding to the central hairpin forms another thermodynamically stable hairpin structure. Its correct folding requires the presence of a ribozyme 3'-terminal sequence and the formation of helix P2. This confirms the ribozyme structure of the pseudoknot type and points to the crucial role of helix P2 in its overall folding. Moreover, we show that the J4/2 region can be specifically cleaved in the presence of selected divalent metal ions in the full-length transcript, but not in a shorter one lacking six 3'-terminal nucleotides, which cannot form the pseudoknotted structure. Thus, a particular RNA conformation around that cleavage site is required for specific hydrolysis, and the J4/2 region seems to be involved in the formation of a general metal ion binding site. Recently, it has been proposed that, in the antigenomic ribozyme, a four nucleotide sequence within the J1/2 region may contribute to the folding pathway, being part of a mechanism responsible for controlling ribozyme cleavage activity. Our study shows that in the genomic ribozyme the central hairpin region may contribute to a similar mechanism, providing a barrier to the formation of an active structure in the ribozyme folding pathway.

Patterns of cleavages induced by lead ions in defined RNA secondary structure motifs.

We have characterized the susceptibility of various RNA bulges, loops and other single-stranded sequences to hydrolysis promoted by Pb2+. The reactivity of bulges depends primarily on the structural context of the flanking base-pairs and the effect of nucleotide present at the 5' side of the bulge is particularly strong. The efficiency of stacking interactions between the bulged residue and its neighbors seems to determine cleavage specificity and efficiency. Hydrolysis of two- and three-nucleotide bulges depends only slightly on their nucleotide composition. In the case of terminal loops, the efficiency of their hydrolysis usually increases with the loop size and strongly depends on its nucleotide composition. Stable tetraloops UUCG, CUUG and GCAA are resistant to hydrolysis, while in some other loops of the GNRA family a single, weak cleavage occurs, suggesting the existence of structural subclasses within the family. A very efficient, specific hydrolysis of a phosphodiester bond in the single-stranded region adjacent to the stem in oligomer 12 resembles highly specific cleavages of some tRNA molecules. The reaction occurs in the presence of Pb2+, but not in the presence of several other metal ions. The Pb(2+)-cleavable RNA domain may be considered another example of leadzyme. The results of Pb(2+)-induced hydrolysis in model RNA oligomers should be useful in interpretation of cleavage patterns of much larger, naturally occurring RNA molecules.

A cell-free yellow lupin extract containing activities of pseudouridine 35 and 55 synthases.

Plant cytoplasmic tyrosine tRNA was pseudouridylated at three different positions: 35, 39 and 55. These pseudouridines were introduced by three different enzymes--pseudouridine synthases. Variants of the Arabidopsis thaliana pre-tRNA(Tyr) were constructed that allow to monitor specifically pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis we have prepared an extract from Lupinus luteus cv. Ventus seeds containing activities of at least psi35 and psi55 synthases. This is the first report describing the preparation of the lupin seed extract that specifically modifies plant pre-tRNA(Tyr) transcribed by T7 RNA polymerase. U35 is converted to psi35 only in an intron-dependent manner, while pseudouridylation of U55 is insensitive to the presence or absence of an intron.

Cleavages induced by different metal ions in yeast tRNA(Phe) U59C60 mutants.

The U59 and C60 residues, which form the strong Pb(II) ion binding site in yeast tRNA(Phe), were systematically mutated, and the effects of individual mutations on cleavages induced by various metal ions were analyzed. It turned out that the presence of C60 is essential for efficient cleavage of the D-loop by Pb(II), Eu(III), and Mg(II)ions. On the other hand, manganese ions were capable of cleaving these mutants with an efficiency similar to that observed for the wild type transcript. Moreover, in all C60 mutants, the main Mn(II) cleavage site was shifted by one phosphate from P16 to P17. Mutations of U59 did not affect so dramatically the efficiency and specificity of the D-loop hydrolysis induced by all studied metal ions. In the G59C60 mutant cleaved by Pb(II) ions, new cuts took place in the T-stem at P63-P65. Also, the C60 mutants were cleaved more strongly at P49 by Pb(II) ions. In G59C60 and A59C60 as well as in all C60 mutants, the Mg(II) and Mn(II) cleavage at P61 was suppressed. Nevertheless, the changes in overall tRNA structure resulting from U59 and C60 mutations were rather subtle. The studied mutants showed S1 and V1 nuclease digestion patterns practically indistinguishable from those observed in the wild type transcript. The metal ions are shown to be well-suited for monitoring the local changes in the structure of the investigated tRNA variants and when used as a set of probes can give a more complete picture of changes that occur in transcripts as a result of a mutation.

Effect of modified nucleotides on structure of yeast tRNA(Phe). 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.

The pseudouridine residues of ribosomal RNA.

Pseudouridine (psi), the most common single modified nucleoside in ribosomal RNA, has been positioned in the small subunit (SSU) and large subunit (LSU) RNAs of a number of representative species. Most of the information has been obtained by application of a rapid primed reverse transcriptase sequencing technique. The locations of these psi residues have been compared. Many sites for psi are the same among species, but others are distinct. In general, the percentage psi in multicellular eukaryotes is greater than in prokaryotes. In LSU RNA, the psi residues are strongly clustered in three domains, all of which are near or connected to the peptidyl transferase center. There is no apparent clustering of psi in SSU RNA. The psi sites in LSU RNA overlap those for the methylated nucleosides, but this is not the case in SSU RNA. There are 265 psi sites known to nucleotide resolution, of which 246 are in defined secondary structures, and 112 of these are in nonidentical structural contexts. All 246 psi sites can be classified into five structural types. Two Escherichia coli psi synthases have been cloned and characterized, one for psi 516 in SSU RNA and one for psi 746 in LSU RNA. The psi 746 synthase recognizes free RNA, but the psi 516 enzyme requires an intermediate RNP particle. Possible functional roles for psi in the ribosome are discussed.

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 tRNA(Phe), 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 tRNA(Met) is also located in the D-loop at p16 and it is stronger than that observed in tRNA(Phe). In initiator tRNA(Met) 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 tRNA(Glu) 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 tRNA(Glu) anticodon arm. Mutation of a single base A37G in the anticodon loop of the transcript drastically reduced the specificity of Mn(II)-induced hydrolysis.

Purification, cloning, and properties of the 16S RNA pseudouridine 516 synthase from Escherichia coli.

Pseudouridine (psi) is commonly found in both small and large subunit ribosomal RNAs of prokaryotes and eukaryotes. In Escherichia coli small subunit RNA, there is only one psi, at position 516, in a region of the RNA known to be involved in codon recognition [Bakin et al. (1994) Nucleic Acids Res. 22, 3681-3684]. To assess the function of this single psi residue, the enzyme catalyzing its formation was purified and cloned. The enzyme contains 231 amino acids and has a calculated molecular mass of 25,836 Da. It converts U516 in E. coli 16S RNA transcripts into psi but does not modify any other position in this RNA. It does not react with free unmodified 16S RNA at all, and only poorly with 30S particles containing unmodified RNA. The preferred substrate is an RNA fragment from residues 1 to 678 which has been complexed with 30S ribosomal proteins. The yield varied from 0.6 to 1.0 mol of psi/mol of RNA, depending on the preparation. Free RNA(1-678) was inactive, as was RNA(1-526) and the RNP particle made from it. 23S RNA and tRNAVal transcripts were also inactive. These results suggest that psi formation in vivo occurs at an intermediate stage of 30S assembly. The gene is located at 47.1 min immediately 5' to, and oriented in the same direction as, the bicyclomycin resistance gene. The gene was cloned behind a (His)6 leader for affinity purification. Virtually all of the overexpressed protein was found in inclusion bodies but could be purified to homogeneity on a Ni2+(-) containing resin. Over 200 mg of pure protein could be obtained from a liter of cell culture. Amino acid sequence comparison revealed the existence of a gene in Bacillus subtilis with a similar sequence, and psi sequence analysis established that B. subtilis has the equivalent of psi 516 in its small subunit rRNA. On the other hand, no common sequence motifs could be detected among this enzyme and the two tRNA psi synthases which have been cloned up to now.

A dual-specificity pseudouridine synthase: an Escherichia coli synthase purified and cloned on the basis of its specificity for psi 746 in 23S RNA is also specific for psi 32 in tRNA(phe).

An Escherichia coli pseudouridine (psi) synthase, which forms both psi 746 in E. coli 23S ribosomal RNA and psi 32 in tRNA(Phe), has been isolated and cloned. The enzyme contains 219 amino acids and has a calculated MW of 24,432 Da. Amino acid sequence comparison with the three other psi synthases that have been cloned to date, two for tRNA and one for 16S RNA, did not reveal any common sequence motifs, despite the catalysis of a common reaction. The gene was cloned behind a (His)6 leader for affinity purification. Upon overexpression, most of the enzyme remained soluble in the cell cytoplasm and could be purified to homogeneity on a Ni(2+)-containing resin. The enzyme reacted with both full-length 23S RNA or a fragment from residues 1-847, forming 1 mol psi/mol RNA at position 746, a normal site for psi. The enzyme has no dependence on Mg2+. The same yield was obtained in 1 mM EDTA as in 10 mM Mg2+, and the rate was faster in EDTA than in Mg2+. Full-length 16S RNA or fragments 1-526 or 1-678, as well as tRNA(Val) transcripts, were not modified in either EDTA or Mg2+. tRNA(Phe) transcripts, however, were modified with a yield of 1 mol psi/mol transcript at a rate in EDTA like that of 23S RNA. Sequencing showed all of the psi to be at position 32, a normal site for psi in this tRNA. Both 23S rRNA psi 746 and tRNA psi 32 occur in single-stranded segments of the same sequence, psi UGAAAA, closed by a stem. Therefore, this synthase may require for recognition only a short stretch of primary sequence 3' to the site of pseudouridylation. This is the first example of a dual-specificity modifying enzyme for RNA, that is, one which is specific for a single site in one RNA, and equally site-specific in a second class of RNA. The essentiality of these psi residues can now be assessed by disruption of the synthase gene.

Purification, cloning, and properties of the tRNA psi 55 synthase from Escherichia coli.

tRNA pseudouridine 55 (psi 55) synthase, the enzyme that is specific for the conversion of U55 to psi 55 in the m5U psi CG loop in most tRNAs, has been purified from Escherichia coli and cloned. On SDS gels, a single polypeptide chain with a mass of 39.7 kDa was found. The gene is a previously described open reading frame, p35, located at 68.86 min on the E. coli chromosome between the infB and rpsO genes. The proposed name for this gene is truB. There is very little protein sequence homology between the truB gene product and the hisT (truA) product, which forms psi in the anticodon arm of tRNAs. However, there was high homology with a fragment of a Bacillus subtilis gene that may produce the analogous enzyme in that species. The cloned gene was fused to a 5'-leader coding for a (His)6 tract, and the protein was overexpressed > 400-fold in E. coli. The recombinant protein was purified to homogeneity in one step from a crude cell extract by affinity chromatography using a Ni(2+)-containing matrix. The SDS mass of the recombinant protein was 41.5 kDa, whereas that calculated from the gene was 37.3. The recombinant protein was specific for U55 in tRNA transcripts and reacted neither at other sites for psi in such transcripts nor with transcripts of 16S or 23S ribosomal RNA or subfragments. The enzyme did not require either a renatured RNA structure or Mg2+, and prior formation of m5U was not required. Stoichiometric formation of psi occurred with no requirement for an external source of energy, indicating that psi synthesis is thermodynamically favored.