Biology subjects

Finzi, L.

Publications and source records attributed to Finzi, L..

3 recordsLinked to original sources

bioRxiv2021

Transcription elongation through lac repressor-mediated DNA loops

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, 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. In support of this, RNA polymerase transcribed a looped segment more slowly than an unlooped segment and paused more briefly at LacI obstacles on positively supercoiled templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase appears to facilitate elongation along topologically-complex, protein-coated templates.

biophysics↗

bioRxiv2021

Negative DNA supercoiling tunes protein-mediated looping

Protein-mediated DNA looping is fundamental to gene regulation and such loops occur stochastically in purified systems. Additional proteins increase the probability of looping, but these probabilities maintain a broad distribution. For example, the probability of lac repressor-mediated looping in individual molecules ranged 0-100%, and individual molecules exhibited representative behavior only in observations lasting an hour or more. Titrating with HU protein progressively compacted the DNA without narrowing the 0-100% distribution. Increased negative supercoiling produced an ensemble of molecules in which all individual molecules more closely resembled the average. Furthermore, in only twelve minutes of observation, well within the doubling time of the bacterium, most molecules exhibited the looping probability of the ensemble. DNA supercoiling, an inherent feature of all genomes, appears to impose time-constrained, emergent behavior on otherwise random molecular activity.

biophysics↗

bioRxiv2021

Nanomechanics of negatively supercoiled diaminopurine-substituted DNA

Single molecule experiments have demonstrated a progressive transition from a B- to an L-form helix as DNA is gently stretched and progressively unwound. Since the particular sequence of a DNA segment influences both base stacking and hydrogen bonding, the conformational dynamics of B-to-L transitions should be tunable. To test this idea, DNA with diaminopurine replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded A:T base pairs. Triple hydrogen bonding stiffened the DNA by 30% flexurally. In addition, DAP-substituted DNA formed plectonemes with larger gyres for both B- and L-form helices. Both unmodified and DAP-substituted DNA transitioned from a B- to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing by DNA stretched and unwound by enzymatic activity. The intramolecular nature and ease of this transition likely prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length indicating it is much more contractile and prone to sharp bends and kinks. However, left-handed DAP DNA was twice as stiff as unmodified L-DNA. Thus, significantly doubly and triply hydrogen bonded segments have very distinct mechanical dynamics at physiological levels of negative supercoiling and tension.

biophysics↗