There are three aspects to this project:1.Analysis of new X-ray structures of the ribosomal 23S RNA fragments bound to protein L11 revealed formation of the mini-triple helices predicted by us in 1994 (the so called R-form, or recombination triplex). These mini-triplexes contain iso-geometric GC:G and CG:C triads of bases. Covariation analysis of the 23S RNA sequences indicates that mutations in the latter triad are consistent with formation of one more homologous triad, UA:U, which so far has not been observed directly.2. Radioprobing studies of the RNA polymerase-DNA-RNA complex revealed new details of the spatial arrangement of the RNA strand with respect to the DNA transcription ?bubble? (in collaboration with Dr. R. Neumann, Clinical Center). Among the novel features of the complex is the three- stranded complex DNA-DNA-RNA at the ?upstream? end of the bubble. The functional role of this structure may be related to stabilization of the transcription elongation complex.3. A new project has been initiated to study the R-form triplex in solution. The oligonucleotides with the non-nucleotide linkers were designed based on our calculations, and synthesized -- this was done to facilitate formation of the intranucleotide triplexes and to avoid aggregation. The R- triplex has geometry different from that of the better known H- triplexes. In particular, all three grooves in the R-form have comparable widths. Therefore, it is important to select specific cations that would be most favorable for this unusual structure. The optimal conditions for formation of R-triplexes in solution were found calorimetrically (in collaboration with Dr. A. Shchyolkina, Inst. Mol. Biol., Moscow, Russia). As expected, LiCl in high concentration is the most effective stabilizer of the R-form (Li has a low specificity and a large hydration shell). These conditions will be used in the NMR measurements (in collaboration with Dr. A. Gronenborn, NIDDK).Z01 BC 10046-04 - DNA folding, Gene regulation, molecular interactions, RNA folding, synthesis chemistry, transcriptional control,
