The in vitro transcription units of bacteriophage φX174. I. Characterization of synthetic parameters and measurement of transcript molecular weights

Abstract

In vitro transcription of bacteriophage replicative φX174 form I DNA by Escherichia coli RNA polymerase holoenzyme is studied under a variety of ionic conditions. Uniformly ^(32)P-labeled product RNA is fractionated by electrophoresis on 2% acrylamide, 0.5% agarose or 7.5 mm-CH_3HgOH-containing, 1.5% agarose slab gels. A large fraction (65 to 70%) of the RNA made during 30 minutes synthesis at 37 °C is present as a heterodisperse population of molecules with a mean molecular weight of 2 to 3 × 10^6. From 30 to 35% of the product RNA is present in several discretely sized bands of molecular weights 1.55, 1.95, 2.35, 3.70, 4.0, 4.15 and approximately 5 × 10^6. All components except the first arise from more than one circumtranscription of φX replicative form I template. Thus specific termination is detected under the conditions studied, but apparently is not totally efficient. A method allowing (α-^(32)P)-labeling of a short (10 to 80 nucleotides) 5′ proximal region of φX174 in vitro RNA is described. Fractionation of 5′ proximally labeled in vitro φX RNA resolves all of the discrete components detected by the uniform labeling technique and, in addition, several smaller components. Approximately 50% of the molecules synthesized are both initiated and terminated specifically; the remaining 50% of the molecules synthesized are present as heterodisperse material presumably arising from either non-specific initiation or termination (or both).