SP6 RNA polymerase stutters when initiating from an AAA… sequence

Abstract

The 16S ribosomal RNA gene of Escherichia coli was placed under the transcriptional control of consensus and modified T7 promoters and a modified SP6 promoter. Both T7 and SP6 polymerases faithfully transcribed the coding sequence (beginning at the +1 position) of each construct, although SP6 polymerase was five-fold more effective than T7 polymerase in initiating with the AAAUUG… sequence. An appreciable fraction of the SP6 transcript molecules contained additional adenosines In the −1, −2, −3, −4, and −5 positions. The transcripts containing additional residues constituted approximately 40–50% of the total SP6 transcription products. Neither the nature nor extent of the additional residues was affected by replacing the pppA 5'-end by pA. Since the identity of the inserted residues does not correspond to the sequence of the template, these additional nucleosides must result from ‘wstuttering’ of the SP6 enzyme at the −1 to +3 positions during initiation of transcription.