Transcriptional Obstacles Modify Repetitive Elongation

Abstract

Transcription is regulated by multiple factors including those in the path of RNA polymerase that can block elongation. Obstacles were produced using the lac repressor protein (LacI) binding to the high affinity O1 sequence in DNA tethers anchored by RNA polymerase at one end. Passage through LacI obstacles was monitored using either tethered particle motion (TPM) without tension on the DNA tether, or magnetic tweezers (MTs) applying sub-piconewton tension. When E. coli RNAP labeled on either the α or β’ subunits encountered LacI, it paused and the tether length remained constant for up to minutes. However, three peculiar behaviors were noted. (i) In the absence of tension, the pausing of RNAPs with either labeling was comparable. In contrast, while RNAP attached to glass via hemagglutinin antigen tags on α subunits paused for longer at LacI when tension opposed rather than assisted transcriptional translocation, RNAP attached to beads via a biotin-labeled β’ subunit paused more briefly at LacI when tension opposed rather than assisted transcriptional translocation. (ii) After pausing at a LacI obstacle with tension opposed to transcription, RNAP often abruptly re-commenced elongation from the promoter. Occasionally, having surpassed an obstacle and reached the end of the template, RNAP abruptly translocated to re-commence transcription from the obstacle. (iii) Occasionally, after reaching the end of the template, RNAPs elongating under assisting tension switched strands to transcribe in the opposite direction against tension opposing transcriptional translocation. Although forces applied to different subunits of the enzyme certainly influenced activity, RNAP translocated rapidly or conserved an interaction with the promoter or an obstacle to perform repeated cycles of elongation. Complete reversal of the direction of elongation occurred at double-stranded ends. Repetition and reversal expand the gamet of transcriptional output.