Role of Deoxyribonucleic Acid-Ribonucleic Acid Hybrids in Eukaryotes

The template requirements of yeast RNA polymerases A (or I) and B (or II) and the nature of the product formed have been investigated. The template activity of native DNA is lost after treatment with endonuclease S1, an enzyme highly specific for single-stranded DNA. Intact double helical DNA does not support RNA synthesis. This activity is not improved by low levels of deoxyribonuclease I. An excellent template is produced by successive treatment of T7 DNA by pancreatic DNase and exonuclease II, indicating that RNA polymerases A (or I) and B (or II) can transcribe a duplex structure containing unpaired gaps. The requirement for single strandedness of the template applies only for the initiation step: internal single-strand gaps of 20 to 50 nucleotides long promote RNA chain initiation, and very long RNA chains susceptible to RNase A are synthesized. During synthesis, the 5'-terminus of the RNA chain is degraded by ribonuclease H, indicating that initiation occurs on unpaired DNA with the transient formation of a short RNA-DNA hybrid. Such a transcription complex is very labile and release of RNA chains occurs continuously during transcription.