Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability

We have constructed a series of plasmid templates that allow T7 RNA polymer ase (RNAP) to be halted at defined intervals downstream from its promoter i n a preserved sequence context. While transcription complexes halted at +3 to +6 are highly unstable, complexes halted at +10 to +14 dissociate very s lowly and gradually lose their capacity to extend transcripts. Complexes ha lted at +18 and beyond dissociate more readily, but the stability of the th ese complexes is enhanced significantly in the presence of the next incomin g nucleotide. Unexpectedly, the stability of complexes halted at +14 and be yond was found to be lower on supercoiled templates than on linear template s. To explore this further, we used synthetic DNA templates in which the na ture of the non-template (NT) strand was varied. Whereas initiation complex es are less stable in the presence of a complementary NT strand, elongation complexes are more stable in the presence of a complementary NT strand, an d the presence of a non-complementary NT strand (a mismatched bubble) resul ts in even greater stability. The results suggest that the NT strand plays an important role in displacing the nascent RNA, allowing its interaction w ith an RNA product binding site in the RNAP. The NT strand may also contrib ute to stabilization by interacting directly with the enzyme. A mutant RNAP that has a deletion in the flexible "thumb" domain responds to changes in template topology in a manner that is similar to that of the wild-type (WT) enzyme, but halted complexes formed by the mutant enzyme are particularly dependent upon the presence of the NT strand for stability. Ln contrast, an N-terminal RNAP mutant that has a decreased capacity to bind single-strand ed RNA forms halted complexes with much lower levels of stability than the WT enzyme, and these complexes are not stabilized by the presence of the NT strand. The distinct responses of the mutant RNAPs to changes in template structure indicate that the N-terminal and thumb domains have quite differe nt functions in stabilizing the transcription complex. (C) 2000 Academic Pr ess.