Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops

Abstract

Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase generates supercoiling that may facilitate passage through such barriers. We monitored E. coli RNA polymerase progress along templates in conditions that prevented, or favored, 400 bp LacI-mediated DNA looping. Tethered particle motion measurements revealed that RNA polymerase paused longer at unlooped LacI obstacles or those barring entry to a loop than those barring exit from the loop. Enhanced dissociation of a LacI roadblock by the positive supercoiling generated ahead of a transcribing RNA polymerase within a torsion-constrained DNA loop may be responsible for this reduction in pause time. In support of this idea, RNA polymerase transcribed 6-fold more slowly through looped DNA and paused at LacI obstacles for 66% less time on positively supercoiled compared to relaxed templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase facilitated elongation along topologically complex, protein-coated templates.

Reciprocating RNA Polymerase batters through roadblocks

“…There are other features that are beyond the scope of this work. The experiments and model presented here neglect torsion although reciprocating movement along a few base-pairs in torsionally anchored templates might generate sufficient torsion to stall RNA polymerase 40,41 . Furthermore, backtrack and recovery cycles are faster upstream of EcoRI than upstream of lac binding sites.…”

“…All DNA fragments for MT experiments were PCR amplicons from plasmid templates pZV_NI_400, pDM_N1_400, pWX_12_400 or pDM_E1_400 40 , single-digoxigenin labeled forward and unlabeled reverse primer pairs (Supplementary Data 1-5), and Q5 Hot Start High-Fidelity 2X PCR Master Mix (New England Biolabs, Ipswich, MA). The transcribed region had the following spacings: Promoter-709 bp-Lac operator-612 bp-Terminator, with an EcoRI binding sequence 5'-GAATTC between the promoter and the operator site, as illustrated in Fig.…”

“…2A, B, 0 pN, GreA-condition). The preparation of DNA templates and their assembly into tethers for beads in microchambers has been described in detail elsewhere 40 .…”

Transcription disrupts DNA-scaffolded bacteriophage repressor complexes

“…Lac repressor is a homo-tetramer with two dimeric DNA binding units and can bind two operators simultaneously, securing a loop in the intervening DNA [23, 24]. Remarkably, even LacI bound to O2 significantly impedes transcription when it is part of a loop [25, 26]. Many transcription factors secure loops between distant sites [27, 28], and others may wrap DNA [29].…”

RNA polymerase efficiently transcribes through DNA‐scaffolded, cooperative bacteriophage repressor complexes

“…Lac repressor is a homotetramer with two dimeric DNA-binding units and can bind two operators simultaneously, securing a loop in the intervening DNA [26,27]. Remarkably, even LacI bound to O2 significantly impedes transcription when it is part of a loop [28,29]. Many transcription factors cooperatively secure loops between distant sites [30,31], and others may wrap DNA [32] or bridge contiguous and/or non-contiguous helices.…”