T7 RNA-POLYMERASE BYPASS OF LARGE GAPS ON THE TEMPLATE STRAND REVEALSA CRITICAL ROLE OF THE NONTEMPLATE STRAND IN ELONGATION

We show that T7 RNA polymerase can efficiently transcribe DNA containi ng gaps from one to five bases in the template strand. Surprisingly, b roken template strands missing up to 24 bases can still be transcribed , although at reduced efficiency. The resulting transcripts contain th e full template sequence with the RNA deleted for the gapped region mi ssing on the template strand. These findings indicate that the end of a downstream template strand can be brought into the polymerase and tr anscribed as ii it were a part of an intact polynucleotide chain by ut ilizing the unpaired nontemplate strand. This, as well as transcriptio n of an intact template strand, relies heavily upon the nontemplate st rand, suggesting that a duplex DNA-binding site on the leading edge of RNA polymerase is required for RNA chain elongation on DNA templates. This work contributes substantially to the emerging picture that the nontemplate strand is an important element of the transcription elonga tion complex.