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

Abstract

1. When RNA polymerase is in excess over DNA, the single-stranded breaks ofDNA can be recognized as initiation sites for the enzyme. On the other hand, stable initiation complexes (resistant to inhibition by heparin) are the most abundant under these conditions. The formation of these complexes needs double-stranded DNA. It seems that RNA sequences rich in cytidine are preferentially synthesized; since rat liver DNA is A+T-rich, the transcription thus appears not to be random with respect to the base composition of DNA. 2. When the template is in excess over the polymerase, the single-stranded gaps ofDNA are preferentially transcribed by rat liver RNA polymerase B, and native DNA regions by Escherichia coli RNA polymerase. 3. With a large excess of DNA over the polymerase, the enzyme activity is markedly inhibited. This inhibition is proportional to the concentration of double-stranded DNA ends, but it also depends on the presence of a contaminant of DNA, removed when DNA is banded in a CsCl gradient. This contaminant could be pclyphosphates. Low concentrations of spermine completely reverse this inhibition, by enhancing the rate of RNA chain elongation. 4. Double-stranded RNA is synthesized in great abundance when RNA polymerase is in excess over native DNA. Besides a majority of symmetrical sequences, stable 'hairpins' can be found. Whereas the synthesis of symmetrical sequences is more prevalent in polymerase excess, it seems that the proportion of stable 'hairpins' in RNA is independent of the polymerase/DNA ratio.