Interaction between Denatured DNA and Ribosomal RNA from Bacteria: Characterisation of DNA Sites Interacting with Ribosomal RNA at Room Temperature

Abstract

The interaction which takes place a t room temperature between bacterial denatured DNA and ribosomal RNA has been studied by filtration on nitrocellulose filters and on Sephadex columns. The optimal conditions for this kind of interaction have been1determined. The melting curves and the effect of ribonuclease treatment are consistent with the hypothesis that in this kind of hybrids short base paired regions are present and unpaired RNA tails remain which are digested by ribonuclease. The average length of base paired regions has been shown to be about 20 nucleotides.The sites on DNA which can interact with RNA a t room temperature and which are located in all studied cases on the transcribed DNA strand (or transcribed regions of both strands), have been shown by saturation experiments to cover a large proportion (0.55O/, in Bacillus steurothermophilus) of bacterial DNA.An approximate calculation of the number of these sites in DNA from B. stearotermophilus, made on the basis of the present results, indicates the existence of one site per 1800 nucleotide pairs.It is well known that ribosomal RNA can anneal with denatured homologous DNA upon incubation a t 40-60" for several hours and that the sites for ribosomal RNA account for about 0.3O/, of bacterial DNA [I -31. Significant hybridization has been obtained also in heterologous mixtures of ribosomal RNA and DNA from several bacteria; this indicates a high degree of sequence homologies among bacterial cistrons for ribosomal RNA [4]. It has been recently demonstrated [5--81 that a different kind of interaction can take place a t room temperature between denatured DNA from some microorganisms and homologous as well as heterologous ribosomal RNA.This type of DNA-RNA complex, for which the name of pseudohybrid has been proposed [S], is partially resistent to ribonuclease ; its formation was first demonstrated by the appearance of a new band in CsCl density gradients. I n previous works [8,5] it has been demonstrated that only one of the two complementary DNA strands is involved in this kind of interaction with ribosomal RNA, and work subsequently performed using DNA from phage a [9] has shown that only the strand which is physioEnzymes. Pancreatic ribonuclease, ribonucleate pyrimidine-nucleotido-2'-transferase (cyclizing) (EC 2.7.7.16); ribonuclease T 1, ribonucleate guaninenucleotide-2'-transferase (cyclizing) (EC 2.7.7.26); deoxyribonuclease, deoxyribonucleate oligonucleotidohydrolase (EC 3.1.4.5).logically transcribed participates to the formation of pseudohybrids.This kind of DNA-RNA interaction has been observed in DNA-RNA mixtures containing DNA from several bacilli but no formation of a pseudohybrid band had been observed when the mixture contained DNA:from Escherichiu coli [8,5].A similar kind of interaction has been shown to take place between denatured DNA and polyguanylic acid [5,6,10].I n phage 1, in which transcription occurs on alternating regions of both DNA strands [ll, 121 the sites interacting with poly G are located on the transcribed region of eac...