Further characterization of bacteriophage T3-induced ribonucleic acid polymerase

Abstract

The size of RNA chains synthesized in vitro by T3 RNA polymerase from a T3 DNA template has been analyzed by polyacrylamide gel electrophoresis under native and denaturing conditions. In uitro, eight discrete size classes of T3 RNAs were observed, designated I to VIII. The apparent molecular weights, estimated from their electrophoretic mobilities, are approximately 6.2, 4.7, 4.0, 2.8, 1.8, 0.9, 0.52, and 0.21 × lo⁶, respectively. In contrast, the late mRNAs isolated from TS-infected Escherichia coli consist of seven discrete size classes of RNA chains ranging in molecular weights between 0.52 to 3.5 × 10". The largest in vitro transcripts, I, II, III, and IV, and the smallest species, VIII, do not seem to have an in uiuo counterpart. The remaining three in vitro transcripts (V, VI, and VII) each appear to migrate in the gel with an apparent molecular weight similar to an in uiuo RNA species. All the major in uitro T3 RNA species are initiated simultaneously and independently with guanosine triphosphate at the 5' terminus and are elongated at the rate of approximately 290 nucleotidesls at 37" under standard in vitro conditions. Furthermore, except for species I and IV, all other in vitro RNA species are synthesized in approximately equimolar amounts; species I and IV approximated one-third and one-half, respectively, of the molar amount of each of the other six species. The nature of the binary complexes formed between the phage T3 RNA polymerase and T3 DNA have been studied. In the absence of all four nucleoside triphosphates, and at or below 0.5 mM Mg²⁺, T3 RNA polymerase forms a measurable complex with T3 [³H]DNA detectable by both Millipore fil-tration and by glycerol gradient centrifugation techniques. Upon addition of all four nucleoside triphosphates, the polymerase. DNA complex initiates synthesis of RNA chains without the addition of any exogenous DNA. Complex formation is highly sensitive to Mg²⁺ and high monovalent salt concentrations; complex formation is virtually abolished at 2 mM or higher Mg^z+ concentration or at 20 mM or higher K+ concentrations. Studies on the stability of the complexes between T3 DNA and T3 RNA polymerase indicate that such complexes are relatively unstable compared to complexes formed with E. coli RNA polymerase and T7 DNA; the halftime of dissociation of T3 RNA polymerase T3 DNA complexes at 37" is approximately 1 min, a value several orders of magnitude lower than complexes formed by E. coli RNA polymerase with T7 DNA.