Structure of the RNA portion of the RNA-linked DNA pieces in bacteriophage T4-infected Escherichia coli cells

Abstract

A method for the isolation of the RNA portion of RNA-linked DNA fragments has been developed. The method capitalizes on the selective degradation of DNA by the 3′ to 5′ exonuclease associated with bacteriophage T4 DNA polymerase. After hydrolysis of the DNA portion, the RNA of RNA-linked DNA is recovered mostly as RNA tipped with a deoxyribomononucleotide and a small fraction as pure RNA. On the other hand, the 5′ ends of RNA-free DNA are recovered mostly as dinucleotides and a small fraction as mononucleotides.

Using this method, we have isolated the primer RNA for T4 phage DNA synthesis. Nascent short DNA pieces were isolated from T4 phage-infected Escherichia coli cells and the 5′ ends of the pieces were dephosphorylated and then phosphorylated with polynucleotide kinase and [γ-³²P]ATP. After selective degradation of the DNA portions, [5′-³²P]oligoribonucleotides (up to pentanucleotide) were obtained with covalently bound deoxymononucleotides at their 3′ ends. More than 40% of the oligoribonucleotides isolated were pentanucleotides with pApC at the 5′-terminal dinucleotide. The 5′-terminal nucleotide of the tetraribonucleotides was AMP, but that of the shorter chains was not unique. The pentanucleotide could represent the intact primer RNA for T4 phage DNA synthesis.