Displacement of parental RNA strands during in vitro transcription by bacteriophage phi 6 nucleocapsids

We have studied the mode of transcription of the three double-stranded RNA segments found in bacteriophage phi 6. Stable transcription intermediates, isolated following in vitro incorporation of nucleoside triphosphates by phi 6 nucleocapsids, were examined by electron microscopy. Specimens were either spread and shadowed or deposited on polylysine film and stained. In either case, branched molecules with one or more single-stranded arms were seen. The single-stranded arm, in all molecules observed, has about half the contour length of one double-stranded arm. The branched molecules are stable in high salt or hot phenol, resistant to proteinase K, but sensitive to RNAase A in high salt, yielding fragments of double-stranded RNA. These results are consistent with a transcription mechanism in which each new transcript displaces one of the parental RNA strands. From the rate of movement of the branch point, we found transcription rates in vitro of similar to or approximately 25 nucleotides per sec at 30 degrees C and 19 nucleotides per sec at 25 degrees C. Based on the spacing between branches in multiply branched molecules, initiation occurs approximately once every 40 sec at 30 degrees C on M or S RNA templates and about 6 times less frequently on L RNA.