Transcription initiation site of phage SP6 RNA polymerase

Abstract

The transcription initiation sites of the phage SP6 initiation sequence mutants containing deletions around the wild-type initiation site were determined. It could be possible to obtain sequencing ladders in high-resolution polyacrylamide-urea gel electrophoresis, using the phage SP6 RNA polymerase under the in vitro transcription conditions where the limiting concentration of a ribonucleotide causes the polymerase to pause long enough and terminate at the positions of the nucleotide. Precise sizing of the transcripts produced by this abortive elongation determined the initiation site of each mutant. When the wild-type position +1 G is changed to C, it still starts at the C. Also the wild-type position +1 G is changed to A, it still starts at the A. But, the mutant containing CCC sequence from -1 to +2 starts transcription at the C at both the wild-type position -1 and +1 with equal frequency.