Transcriptional block caused by a negative supercoiling induced structural change in an alternating CG sequence

Abstract

Using supercoiled plasmids containing a (CG) 16 sequence downstream of a promoter, it is shown that purified E. coli RNA polymerase can transcribe through the sequence when it is in the B helical form. However, the polymerase together with its nascent transcript is blocked at the boundary of the CG sequence proximal to the promoter when the template is negatively supercoiled to flip the CG sequence to the left-handed Z-form. S1 nuclease mapping of in vivo transcripts from an E. coli gyrase temperature-sensitive mutant harboring the plasmids indicates that the bulk of the transcripts at either permissive or nonpermissive temperatures can proceed through the CG sequence, suggesting that the sequence is normally in the B helical form in vivo. The almost total blockage of transcription in vitro by the (CG) 16 sequence in a highly negatively supercoiled DNA is not observed for a d(CA) 21 · d(TG) 21 insert.