Role of DNA in RNA synthesis. XI. Selective transcription of λ DNA segments in vitro by RNA polymerase of Escherichia coli

In vitro transcription of native DNA isolated from mature bacteriophage λ was studied using highly purified preparations of DNA-dependent RNA polymerase isolated from E. coli W. Half-length segments of sheared λ DNA were separated by density-gradient centrifugation, and the RNA polymerase products synthesized on the whole λ DNA template and on each of its separated halves were characterized with regard to their nearest-neighbor nucleotide frequencies, base composition, average chain-length, sedimentation velocity, and ability to anneal with specific segments of the template. The priming efficiencies of the λ DNA halves were compared, and the influence of certain alterations in the secondary or tertiary structure of the λ DNA template on the RNA products formed in vitro was examined. These studies indicate that transcription of native λ DNA by the E. coli polymerase in vitro is not random; specific template regions present predominantly on the AT-rich (right) half of linear λ DNA are preferentially transcribed throughout the duration of in vitro RNA synthesis. Denaturation of the λ DNA template results in elimination of selective copying. Neither free cohesive ends nor linearly intact DNA are essential for the selection mechanism.