Transcription bypass or blockage at single-strand breaks on the DNA template strand: effect of different 3' and 5' flanking groups on the T7 RNA polymerase elongation complex

Abstract

We have studied the effects of single-strand breaks present on the template strand during T7 RNA polymerase transcription elongation. A synthetic DNA template with a T7 promoter was designed to contain a one-nucleotide gap at a defined location on the template strand. This gap, surprisingly, was efficiently bypassed by T7 RNA polymerase during transcription elongation, and the full-length transcript (FLT37) generated from the bypass event was shortened by one nucleotide compared to the full-length transcript (FLT38) generated from an intact, unbroken template strand. FLT37 did not contain any nucleotide insertions opposite to the gap, so that the RNA sequence downstream from the gap, although accurately transcribed, contained a single base deletion compared to FLT38. This, to our knowledge, is the first demonstration that the continuity of the DNA template strand is not a necessary requirement for DNA-dependent RNA polymerase transcription elongation. DNA templates with different 3' and 5' termini at the single-strand break site were also investigated in this study. One of these templates, 1/3P-4P, which contained 3'- and 5'-phosphoryl termini at the break site, efficiently blocked T7 RNA polymerase. A single phosphoryl group present on either the 3' or the 5' terminus of the break site did not efficiently block RNA polymerase progression, suggesting that the blockage observed with template 1/3P-4P is due to the repulsion between the two phosphoryl termini in the vicinity of the polymerase active site.