The influence of RNA and DNA template structures during transcript elongation by RNA polymerases.

It was previously thought that elongating Escherichia coli transcription ternary complex consists of an RNA polymerase molecule enclosing 17 +/- 1 melted bases (bubble) of the template DNA and a 12-base-pair RNA-DNA hybrid ("transcription bubble paradigm"). Recent evidence suggests that ternary elongation complexes are heterogeneous and possibly vary in bubble size and length of RNA-DNA hybrid. We used a new type of assay to address the relative contributions of bubble size, secondary structure of RNA and RNA-DNA hybrid length during elongation. Synthetic RNA-DNA bubble duplexes are assembled in vitro. RNA structure 5' to the RNA-DNA hybrid, hybrid length and bubble size are systematically changed. The relative efficiency of E. coli and T7 RNA polymerases to elongate RNA primer is quantitated. RNA elongation was high (approximately 22-30%) when a stable hairpin was present towards the 5' end of the primer. Efficiency of elongation was lower for RNA primers without hairpins. Hairpin RNAs with presumed RNA-DNA hybrids of 3-7 bp were efficiently elongated compared to hairpins that presumably form 10bp hybrids. Preformed bubbles of different sizes (2,5 or 20 bases) were functional in all cases where elongation was moderate or high. We concluded that RNA secondary structure plays a dominant role compared to hybrid length or bubble size in determining efficient elongation by RNA polymerases.