Nucleic Acids Research

Molecules of single-stranded ribosomal RNA and double-stranded replicative form of phage f2 RNA ( dsRNA ) adopt a compact form in solutions, containing sufficiently high concentrations of salt ( NaCl ) and polymer ( PEG ). However, only in the case of native dsRNA molecules the compact particles are characterized by a regular internal structure, which accounts for the appearance of an intense positive band in CD spectra. Heating or acidification of PEG-containing solutions of dsRNA leads to the disappearance of the intense positive CD band, which results from the "destruction" of the regular internal structure of compact particles. Comparison of properties of ONA and dsRNA compact particles formed in PEG-containing water-salt solutions suggests the existence of similar mechanisms of compactization of double-stranded polynucleotides. INTRODUCTION It has been shown that native double-stranded DNA molecules in watersalt solutions, containing poly(ethyleneglycol) ( PEG ) can adopt a compact form ( the so-called V'-form ), which is characterized by a regular spatial arrangement of DNA chains. Due to a specific type of ordering of DNA chains within compact particles an intense negative band at 260-270 nm appears in 1-3 4 their CD spectra . It is known also that a non-intercalating antibiotic distamycin A, when complexed with DNA prior to compactization, does not seem to change the mode of DNA packing in compact particles, although in this case an intense positive band at 265-270 nm ( instead of a negative one ) appears in CD spectra. Despite the possibility of theoretical explanations of the appearance of intense bands with different signs in CD spectra ' , the correlation between the secondary structure of polynucleotides, forming compact particles, and the type of their CD spectra is not fully understood. In the present investigation we compared optical and X-ray diffraction characteristics of compact particles, formed from two types of hifch molecular weight RNA: a replicative double-stranded form of phage f2 RNA ( dsRNA ) and © Information Retrieval Limited 1 Falconberg Court London W1V5FG England 1533 Nucleic Acids Research single-stranded tibosomal RNA ( rRNA ). dsRNA, being similar to DNA in some structural parameters, belongs in contrast to DNA not to the B, but to the A family of polynucleotides. MATERIALS AMD METHODS *) 7 To prepare dsRNA a logarithmically-growing culture of E.coli AB 105 in tryptone-yeast extract medium supplemented with L-methionine ( 50AVg/ml ), o was infected with phage f2 sus II ( 10-20 plague-forming units per bacterium ) and cultivated overnight at 34 C on a Dubnoff shaker. DNA and ds RNA were 9 extracted according to Marmur . To remove host nucleic acids crude RNA, dissolved in O.I M NaCl + 10 mM MgCl2 ( about 5 mg/ml of nucleic acids ), was treated with pancreatic DNAse and RNAse ( IO/U-g/ml of each ) for 30 min at room temperature with stirring. The enzymes were inactivated by adding bentonite ( I mg/ml ) and sodium dodecyl sulfate ( 0.1% ), followed by phenol treatment. Degraded host nucleic acids were removed by repeated precipitation 9 with isopropanol as indicated . Traces of polysaccharidic material were removed using the potassium phosphate-2-methoxyethanol extraction step as described by Kirby. The pure dsRNA, dissolved in O.I SSC, had a melting temperature T = 87°C, while no change of absorbance was observed upon heating between 25 and 70 C; the mean sedimentation coefficient s was 10.6. According to w electron-microscopic observations ( Fig.I ) the preparation of dsRMA consisted mainly of double-stranded molecules with the mean length of ( which corresponds to molecular weight of approximately 2x10 daltons ) and also of a small amount of shorter molecules. When examined by acrylamide gel electrophoresis the dsRNA was also shown to consist of integral duplex phage genomes ( molecular weight <~ 2.2x10 daltons), accompanied by small amounts of shorter molecules, forming discrete bands at higher elec12 trophoretic mobilities Preparation of RNA from rat liver polysomes was kindly given to us by E.V. Brikina ( Institute of Medical Biology, AMS USSR ). The preparation consisted of two fractions with sedimentation coefficients of I8S and 28S. Heat denatured dsRNA was prepared by heating a water-salt solution of dsRNA ( 5xI0~ M phosphate buffer + IO~ M EDTA; C ~ 20/VUg/ml ) for 10 min at 100 C. After quick cooling of this solution in an ice bath the remaining __ dsRNA was prepared in the Inst itute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague. 1534 Nucleic Acids Research