RNA Polymerase primed DNA synthesis in T7 Bacteriophage system

Abstract

DNA replication initiation is divided into three steps: Recognition of the replication origin by the initiator protein, melting of the duplex DNA, and synthesis of RNA primer. Primer synthesis is required as there are no known prokaryotic DNA polymerases that can carry out DNA synthesis de novo. In the T7 bacteriophage system and in mitochondrial DNA, the RNA polymerase catalyzes all three steps to initiate DNA replication. The primary replication origin in the T7 genome has two T7 RNA polymerase promoters separated by 52 bp sequence and followed by an AT rich region. There is almost nothing known about the mechanism of RNA‐primed DNA synthesis, including the length of the RNA primer synthesized by T7 RNA polymerase, the mechanism of switching from transcription to DNA synthesis, and why these events occur only at specific promoters in the T7 genome. Whether specific interactions exist between the two polymerases is also not known. Our results suggest that on short DNA substrates, one of the two promoters at the replication origin is sufficient for initiation of DNA synthesis. The RNA polymerase synthesizes primers of 8–12 nucleotide length that are utilized by the DNA polymerase. In the T7 bacteriophage system, this is the critical length at which the transition from transcription initiation to elongation occurs. Our results also show that an actively transcribing RNA polymerase is necessary for the primers to be utilized by the DNA polymerase.Source of research support: NIH