Physical interference between escherichia coli RNA polymerase molecules transcribing in tandem enhances abortive synthesis and misincorporation.

Abstract

Transcription initiation is accompanied with iterative synthesis and release of short transcripts. The molar ratio of enzyme to template was found to be critical for the amounts and distribution of the abortive products synthesized by Escherichia coli RNA polymerase from several promoters. At a high ratio abortive synthesis of 4-8 nt were enhanced at thelambda P R promoter. Removing excess RNA polymerase just before initiation, achieved by washing immobilized transcription complexes, prevented this enhancement. At this high ratio synthesis of an unexpected 6 nt transcript was enhanced when the enzyme stalled at position +32, but not when it stalled at position +73. This transcript had misincorporations at its fifth and sixth positions, probably due to slippage. Hydroxyl radical footprinting of the complex stalled at +32 in the presence of excess enzyme showed that more than one molecule of RNA polymerase was tandemly bound to the same DNA. These results suggest that: (i) when RNA polymerase molecules are tandemly transcribing the same DNA, transient collisions enhance abortive synthesis by the trailing molecule; (ii) when the leading polymerase stalled in the initially transcribed region blocks progression of the trailing polymerase, the latter can commit misincorporations, probably due to slippage synthesis.