Characterization of double-stranded ribonucleic acid sequences present in the initial transcription products of rat liver chromatin

Abstract

At low ionic strength and with a low exogenous RNA polymerase/DNA ratio, rat liver chromatin directs the synthesis in vitro of RNA sequences rich in double-stranded segments. All the transcripts contain at least one double-stranded sequence. Most of the double-stranded segments are formed by intramolecular base-pairing of inverted complementary sequences separated by a single-stranded loop. They are heterogeneous in size, 35-45% of them being more than 80 nucleotides long. They contain 61-64% G+C, whether synthesized by rat liver RNA polymerase (form B) or Escherichia coli RNA polymerase. The largest double-stranded sequences are found in the largest transcripts, and are the most thermostable. The fidelity of base-matching is better in double-stranded transcripts synthesized on rat liver chromatin by homologous polymerase than in those synthesized on it by a bacterial polymerase, or in those synthesized by either of the two polymerases on pure DNA.

Transcription of rat liver deoxyribonucleic acid in vitro at low ionic strength

“…The different DNA samples I used were all prepared starting from rat liver DNA purified from nuclei as previously described (Pays, 1976), but with extensive RNAase A and proteinase K digestions instead of phenol extractions; this DNA preparation is referred to as 'control' DNA, or 'DNA containing gaps'. Its mean molecular weight, determined by analytical centrifugation (Studier, 1965), was 107 in neutral buffer, and 2.5 x 106 in denaturing conditions.…”

“…The control was run in the same way but without RNAase. Material resistant to RNAase in high salt was considered as double-stranded (Pays, 1976 2-fold decrease in RNA synthesis, compared with that at the optimum 0.1 mM concentration. However, I used these conditions because they gave rise to the synthesis of a large amount of dsRNA on chromatin (Pays, 1976).…”

“…The transcription assays were as described (Pays, 1976). Unless specified, they always contained the same amount of polymerase: either I,ug of E. coli polymerase (80 % pure, as revealed by polyacrylamide-gel electrophoresis; Burgess, 1969) (1977).…”

“…A large fraction of these segnents seem to be formed by intramolecular base-pairing (Pays, 1977). The synthesis of these dsRNA molecules could be related to specificity of transcription, since it is more prevalent on chromatin than on deproteinized DNA, and with homologous than with bacterial RNA polymerase.…”

“…I have found (Pays, 1976(Pays, , 1977) that, at low ionic strength in vitro, rat liver RNA polymerase B (EC 2.7.7.6) directs the synthesis on chromatin or DNA of short RNA sequences containing a high proportion of double-stranded segments (dsRNA). A large fraction of these segnents seem to be formed by intramolecular base-pairing (Pays, 1977).…”