Studies on T3 induced ribonucleic acid polymerase. IV. Transcription of denatured deoxyribonucleic acid preparations by T3 ribonucleic acid polymerase

Transcription of denatured and naturally occurring single stranded DNA preparations by T3 induced RNA polymerase is investigated. Although T3 RNA polymerase possesses a highly specific template requirement and will copy only T3 DNA when native double stranded template is used, the enzyme will transcribe a variety of single stranded DNAs (heat denatured T3, T7, calf thymus, and naturally occurring single stranded circular DNAs like phiX 174 and fd) at a markedly reduced rate. The product formed in a denatured DNA primed reaction is a DNAxRNA hybrid with > 80% of RNA chains initiated with pppG. Some chains (approximately 10 to 15%) are initiated with pppA. The average chain length of RNA produced is considerably smaller (200 to 300 nucleotides) than that obtained in a native T3 DNA directed reaction (5000 to 6000 nucleotides). With denatured DNA as template, T3 RNA polymerase is also able to carry out de novo synthesis of poly(A) chains. This reaction requires high concentrations of ATP and is inhibited by the presence of relatively low concentrations of other ribonucleoside triphosphates. Poly(A) chains formed are not attached to the template DNA, contain ATP at the 5' terminus, and are heterogeneous in size ranging in molecular weight from about 100,000 to 2.1 x 10⁶. These reactions catalyzed by T3 RNA polymerase are analogous to those carried out by Escherichia coli RNA polymerase and suggest that the high degree of template specificity of T3 RNA polymerase residues both in the enzyme and in the secondary structure of the template DNA used to direct the polymerase reaction.