We present an approach for studying the role of complementary nucleic
acid interactions in transcription elongation by E. coli RNA polymeras
e (RNAP). The method involves in vitro reconstitution of a catalytical
ly active elongation complex (EC) by the addition of RNAP to a single-
strand DNA oligonucleotide containing the preannealed RNA primer, foll
owed by incorporation of the complementary nontemplate DNA oligonucleo
tide. In all parameters tested, the reconstituted complex is indisting
uishable from normal EC obtained by promoter-specific initiation. Usin
g RNA primers of different lengths, which were fully or partially comp
lementary to the DNA, we determined the minimal transcript length and
the degree of its template pairing that is required to stabilize prote
in/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 ne
cessary for the high processivity of EC during RNA chain elongation.