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A study was conducted to explore the specificity and function of interaction between the tumor suppressor protein p53 in the yeast three-hybrid system (Y3H) and RNA. The findings prove that p53 binds RNA with little sequence specificity, RNA binding has the potential to regulate DNA binding, and RNA-p53 interactions could be regulated by acetylation of the p53 C-terminus.

A study aims to test the hypothesis that placement of fixed charges near one face of the DNA double helix could induce DNA binding by a purely electrostatic mechanism. The findings are consistent with an electrostatic model of DNA binding in predicted directions.

We have recently identified an RNA aptamer for the transcription factor NF-[kappa]B p50 homodimer [[(p50).sub.2]], which exhibits little sequence resemblance to the consensus DNA target for [(p50).sub.2], but binds [(p50).sub.2] with an affinity similar to that of the optimal DNA target. We describe here the 2.45-[Angstrom] resolution x-ray crystal structure of the p50 RHR/RNA aptamer complex. The structure reveals that two RNA molecules bind independent of each other to the p50 N-terminal lg-like domains. The RNA secondary structure is comprised of a stem and a stem-loop separated by an internal loop folded into a kinked helix because of the cross-strand stacking of three internal loop guanines. These guanines, placed at the edge of the 3' helix, together with the major groove of the irregular 3' helix, form the binding surface for p50. Each p50 monomer uses the same surface to recognize the distorted RNA major groove as observed in the [kappa]B DNA/p50 RHR complex, resulting in strikingly similar interfaces. The structure reveals how the aptamer specifically selects p50 and discriminates against p65. We also discuss the physiological implications of RNA binding by [(p50).sub.2].

In vitro-selected RNA aptamers are potential inhibitors of disease-related proteins. Our laboratory previously isolated an RNA aptamer that binds with high affinity to human transcription factor NF-[kappa]B. This RNA aptamer competitively inhibits DNA binding by NF-[kappa]B in vitro and is recognized by its target protein in vivo in a yeast three-hybrid system. In the present study, yeast genetic selections were used to optimize the RNA aptamer for binding to NF-[kappa]B in the eukaryotic nucleus. Selection for improved binding to NF-[kappa]B from RNA libraries encoding (i) degenerate aptamer variants and (ii) sequences present at round 8 of 14 total rounds of in vitro selection yielded RNA aptamers with dramatically improved in vivo activity. Furthermore, we show that an in vivo-optimized RNA aptamer exhibits specific 'decoy' activity, inhibiting transcriptional activation by its NF-[kappa]B target protein in a yeast one-hybrid assay. This decoy activity is enhanced by the expression of a bivalent aptamer. The combination of in vitro and in vivo genetic selections was crucial for obtaining RNA aptamers with in vivo decoy activity.

Research has been conducted on the DNA bending. The effect of peptide sequence on DNA bending and the hypothesis that the structural asymmetry of peptides produces electrophoretic anomalities in the DNA bending absence have been tested and are reported.

Results point out that both the electrophoretic phasing and fluorescence resonance energy transfer data are consistent with the basic leucine zipper (bZIP) peptide-induced DNA bending model. Data suggest that the trajectory of DNA responds to asymmetric changes in local charge density as a result of electrostatic character of the amino acids.

An RNA aptamer is described that binds to the p50 homodimer form of human transcription factor NF-kappaB. RNA aptamers could be used to inhibit the function of DNA-binding proteins.

A small RNA aptamer that binds with nanomolar affinity to human transcription factor NF-kB was isolated by in vitro selection. Selected RNAs have 31-nucleotide core domains that were necessary and sufficient for NF-kB binding. The results showed that the binding of the RNA aptamer blocks the ability of NF-kB to bind duplex DNA. This expression of the aptamer structure within heterologous nuclear transcripts provide a new method to inhibit NF-k function in vivo.

The role of electrostatic effects on DNA bending by bZIP proteins was investigated. Results revealed that the yeast bZIP transcription factor GCN4 does not induce DNA bending in vitro. GCN4 derivatives showed that their direction and extent of DNA bending are a linear function of the charges of amino acids adjacent to the basic domain of the protein. This relation held over the charge range +6 to -6.

Electrophoretic gel mobility shift titration studies of oligonucleotide-directed triple helix formation in the purine motif via binding of guanine-rich oligonucleotides to duplex DNA confirm that physiological concentrations of Na+ and K+ inhibit triple helix formation. Competing equilibria, in which guanine-rich oligonucleotides form aggregates involving guanine quartets, lead to triplex inhibition. Physiological polyamines reverse the K+-induced inhibition of triplexes.

Homopurine target domains exhibit duplex DNA recognition of oligonucleotide-directed triple helix synthesis. Oligonucleotide probes simultaneously detect homopurine target domains by using DNA strands. Electrophoretic mobility and chemical protection methods are used for detecting the nature of the process of DNA recognition. The investigation reveals the presence of target site with three domains filled with purines.

A study was conducted to develop an artificial and functional repressor/operator interaction that does not require peptides. The triple-helical DNA complexes studied were found to inhibit T7 RNA polymerase initiation but were not observed to inhibit transcription elongation. Further results also suggest that nucleic acids or ribonucleoproteins may influence some sequence-specific DNA interactions that control transcription initiation.

The functional consequences of the inhibition of DNA binding to the eukaryotic transcription factor Sp1 by a site-specific triple helical DNA complex was studied. In particular, the effect of pyrimidine motif triple-helical complexes positioned upstream of the TATA box on the transcription initiation from eukaryotic promoters in a cell-free system was analyzed. The results indicate that the complexes repress transcription by blocking promoter DNA assembly into initiation complexes rather than by occluding Sp1 binding.