Physical Mapping of Transcribing Regions on Coliphage fd DNA by the Use of Restriction Endonucleases
In an RNA-synthesizing system consisting of Escherichia coli RNA polymerase holoenzyme and doubly closed replicative form (RF-I) DNA of phage fd, several species of RNA with unique starting sequences and size are transcribed from the “minus” strand of the template. Transcription of an RNA species starting with pppA proceeded to the size of about 26S and terminated, and the other RNA species starting with pppG had sizes of about 10S, 13S, and 17S (16). The results suggested that RF-I DNA provides sets of specific sites for initiation and termination of RNA transcription. As the transcription proceeds in the same direction, it is likely that the template provides a single termination site, so that RNA chains initiated at different loci proceed to the termination site, at which RNA synthesis ceases. However, there was no direct evidence supporting this model. In this chapter, experiments are described in which RF-I DNA was cleaved by endonucleases with cleavage-site specificities, and the template activity of the resulting unique fragments and the size of synthesized products were analyzed. As a result, we could map the approximate sites at which initiation and termination took place on RF-I DNA.
KeywordsMigration Sucrose Phenol Sedimentation Electrophoresis
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