Role of DNA Bubble Collapse in Initiation and the Transition to Elongation-A Common Mechanism in all RNA Polymerases

Abstract

The well-studied RNA polymerases fall into two distinct classes: the “single subunit” family represented by bacteriophage T7, mitochondrial, and chloroplast RNA polymerases and the “multi-subunit” bacterial and eukaryotic enzyme family. All RNA polymerases are characterized by a relatively unstable initial phase (abortive cycling) and by a significant structural rearrangement associated with the transition to stable elongation. Our previous studies with the single subunit T7 RNA polymerase have established a key role for downstream DNA bubble collapse in the instability of halted initiating complexes. We now extend these studies to the initially transcribing complexes of sigma70-E. Coli RNA polymerase transcribing on variants of the T5 N25 promoter. Specifically, we have studied the role of downstream DNA bubble collapse in initially transcribing complexes by designing partially single stranded DNA constructs with the nontemplate absent at the downstream edge of the transcription bubble. As in our earlier studies, the current results show that collapse of the downstream end of the transcription bubble contributes to instability of a halted complex. This increases turnover and hence the amount of abortive transcripts. Transcription to elongation on these constructs show 18-23mer RNA products. We propose that absence of the collapse of the upstream end of the transcription bubble impairs proper displacement of the 5’end of RNA into the exit channel, leading to unstable complexes. This parallels our earlier model in the T7 system in which upstream bubble collapse (−4 to +2) facilitates displacement of the RNA from the hybrid (impairment of collapse leads to release of 11-13mer RNAs) enabling the transition to elongation.