Crucial role of the RNA:DNA hybrid in the processivity of transcription

We present an approach for studying the role of complementary nucleic acid interactions in transcription elongation by E. coli RNA polymerase (RNAP). The method involves in vitro reconstitution of a catalytically active elongation complex (EC) by the addition of RNAP to a single-strand DNA oligonucleotide containing the preannealed RNA primer, followed by incorporation of the complementary nontemplate DNA oligonucleotide. In all parameters tested, the reconstituted complex is indistinguishable from normal EC obtained by promoter-specific initiation. Using RNA primers of different lengths, which were fully or partially complementary to the DNA, we determined the minimal transcript length and the degree of its template pairing that is required to stabilize protein/nucleic acid interactions in EC to the high level characteristic of normal transcription, Our data show that a hybrid at least 9 nt long, formed between the template DNA and 3'-proximal RNA transcript, is necessary for the high processivity of EC during RNA chain elongation.