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Pbz+-induced hydrolysis of RNase P RNAs from Escherichia coli and the thermophilic eubacterium Thermus thermophilus HB8 revealed one prominent site-specific cleavage in the two RNAs and several minor cleavage sites in structurally corresponding regions of both RNAs. Data presented here and in a previous study [Kazakov, S. & Altman, S. (1991) Proc. Natl Acad. Sci. USA 88, 9193 -91971 provide evidence for several ubiquitous metal-ion-binding sites in eubacterial RNase P RNA subunits. With the I: thermophilus RNase P RNA, susceptibility to Pb2+-induced strand scission at the most prominent site was hypersensitive at the temperature of highest enzyme activity (55 "C). Pb" hydrolysis at this site was strongly reduced at a temperature of 37 "C, where processing is also inefficient. For E. coEi RNase P RNA, specific changes in the lead hydrolysis pattern were observed due to the presence of excess tRNA. Thus, Pb2+-induced hydrolysis seems suitable to sense different conformations of RNase P RNAs. The i ? thermophilus RNase P RNA, in particular, displayed significant processing activity after severe fragmentation by Pb2+, and therefore appears to be suited for reconstituting an active enzyme from RNA subfragments.The catalytic RNA subunits of eubacterial RNase P enzymes are sufficient in vitro to generate the mature 5' termini of precursor tRNAs (Guerrier-Takada et al., 1983). Cleavage of pre-tRNAs requires divalent cations, Mg" being most efficient (Guerrier-Takada et al., 1986. Though the essential role of divalent cations appears to be catalytic , they are likely to be further involved in maintaining spatial arrangements in RNase P RNA and RNA substrates (Holbrook et al., 1978), as well as in stabilizing enzyme-substrate complexes (Hardt et al., 1993). Metal-ion-induced cleavage of Escherichia coli RNase P RNA has been demonstrated to occur at five principal sites, and several metal ion cofactors such as Mn2+, Ca2+, S f ' and Ba2+, as well as metal ion inhibitors of RNase P RNA such as Zn", Coz+, Ni2+ and Cu2+, were shown to promote specific hydrolysis .The specific cleavage of yeast tRNAPh" between U17 and G18 by lead ions (Werner et al., 1976: Brown et al., 1983and 1985Rubin and Sundaralingam, 1983: Krzyzosiak et al., 1988; Behlen et al., 1990) has been exploited as a sensitive probe for the tertiary folding of tRNA variants (Behlen et al., 1990: Pan et al., 1991: Pan and Uhlenbeck, 1992 Dichtl et al., 1993). Beside highly specific Pbz+-induced hydrolysis due to the presence of tight metal-ion-binding sites, cleavages of lower intensity and specificity are observed at higher Pb2+ concentrations. It has been shown for several 5s rRNA molecules that cleavages occur mainly in singlestranded RNA regions, whereas helical regions are highly resistant to hydrolysis (Ciesiolka et al., 1992 a, b).Here we have analyzed lead-ion-induced hydrolysis of RNase P RNAs from E. coli and Thermus thermophilus HB8, an extreme thermophilic eubacterium (Oshima and Imahori, 1974). Preparation of RNAs E. coli RNase P RNA was transcri...

The p53 protein is one of the major factors involved in cell cycle control, DNA repair, and induction of apoptosis. We determined the secondary structure of the 5'-terminal region of p53 mRNA that includes two major translation initiation codons AUG1 and AUG2, responsible for the synthesis of p53 and its N-truncated isoform ΔN-p53. It turned out that a part of the coding sequence was involved in the folding of the 5' untranslated region for p53. The most characteristic structural elements in the 5'-terminal region of p53 mRNA were two hairpin motifs. In one of them, the initiation codon AUG1 was embedded while the other hairpin has been earlier shown to bind the Mdm2 protein. Alternative mechanisms of p53 mRNA translation initiation were investigated in vitro using model mRNA templates. The results confirmed that initiation from AUG1 was mostly cap-dependent. The process was stimulated by a cap structure and strongly inhibited by a stable hairpin at the template 5' end. Upon inhibition, the remaining protein fraction was synthesized in a cap-independent process, which was strongly stimulated by the addition of a cap analogue. The translation initiation from AUG2 showed a largely cap-independent character. The 5' cap structure actually decreased initiation from this site which argues against a leaky scanning mechanism but might suggest the presence of an IRES. Moreover, blocking cap-dependent translation from AUG1 by the stable hairpin did not change the level of initiation from AUG2. Upon addition of the cap analogue, translation from this site was even increased.

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.

Recently, we have determined the secondary structure of the 5'-terminal region of p53 mRNA that starts from the P1 transcription initiation site and includes two major translation initiation codons responsible for the synthesis of p53 and ΔNp53 isoform. Here, we showed that when this region was extended into 5' direction to the P0 transcription start site, the two characteristic hairpin motifs found in this region were preserved. Moreover, the presence of alternatively spliced intron 2 did not interfere with the formation of the larger hairpin in which the initiation codon for p53 was embedded. The impact of the different variants of p53 5'-terminal region, which start at P0 or P1 site and end with the initiation codon for p53 or ΔNp53, on the translation of luciferase reporter protein was compared. Strikingly, the efficiency of translation performed in rabbit reticulocyte lysate differed by two orders of magnitude. The toe-printing analysis was also applied to investigate the formation of the ribosomal complex on the model mRNA constructs. The relative translation efficiencies in HeLa and MCF-7 cells were similar to those observed in the cell lysate, although some differences were noted in comparison with cell-free conditions. The results were discussed in terms of the role of secondary structure folding of the 5'-terminal region of p53 mRNA in translation and possible modes of p53 and ΔNp53 translation initiation.

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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.