When push comes to shove - RNA polymerase and DNA-bound protein roadblocks

In recent years, transcriptional roadblocking has emerged as a crucial regulatory mechanism in gene expression, whereby other DNA-bound obstacles can block the progression of transcribing RNA polymerase (RNAP), leading to RNAP pausing and ultimately dissociation from the DNA template. In this review, we discuss the mechanisms by which transcriptional roadblocks can impede RNAP progression, as well as how RNAP can overcome these obstacles to continue transcription. We examine different DNA-binding proteins involved in transcriptional roadblocking and their biophysical properties that determine their effectiveness in blocking RNAP progression. The catalytically dead CRISPR-Cas (dCas) protein is used as an example of an engineered programmable roadblock, and the current literature in understanding the polarity of dCas roadblocking is also discussed. Finally, we delve into a stochastic model of transcriptional roadblocking and highlight the importance of transcription factor binding kinetics and its resistance to dislodgement by an elongating RNAP in determining the strength of a roadblock.Nan Hao, Alana J. Donnelly, Ian B. Dodd, Keith E. Shearwi

The pIT5 Plasmid Series, an Improved Toolkit for Repeated Genome Integration in E. coli

Published: June 30, 2021We describe a new set of tools for inserting DNA into the bacterial chromosome. The system uses site-specific recombination reactions carried out by bacteriophage integrases to integrate plasmids at up to eight phage attachment sites in E. coli MG1655. The introduction of mutant loxP sites in the integrating plasmids allows repeated removal of antibiotic resistance genes and other plasmid sequences without danger of inducing chromosomal rearrangements. The protocol for Cre-mediated antibiotic resistance gene removal is greatly simplified by introducing the Cre plasmid by phage infection. Finally, we have also developed a set of four independently inducible expression modules with tight control and high dynamic range which can be inserted at specific chromosomal locations.Nan Hao, Qinqin Chen, Ian B. Dodd, and Keith E. Shearwi

RNA polymerase pausing at a protein roadblock can enhance transcriptional interference by promoter occlusion

Convergent promoters exert transcriptional interference (TI) by several mechanisms including promoter occlusion, where elongating RNA polymerases (RNAPs) block access to a promoter. Here, we tested whether pausing of RNAPs by obstructive DNA-bound proteins can enhance TI by promoter occlusion. Using the Lac repressor as a 'roadblock' to induce pausing over a target promoter, we found only a small increase in TI, with mathematical modelling suggesting that rapid termination of the stalled RNAP was limiting the occlusion effect. As predicted, the roadblock-enhanced occlusion was significantly increased in the absence of the Mfd terminator protein. Thus, protein roadblocking of RNAP may cause pause-enhanced occlusion throughout genomes, and the removal of stalled RNAP may be needed to minimize unwanted TI.Nan Hao, Michael T. Crooks, Adam C. Palmer, Ian B. Dodd, Keith E. Shearwi

Analysis of infection time courses shows CII levels determine the frequency of lysogeny in phage 186

Published: 29 September 2021Engineered phage with properties optimised for the treatment of bacterial infections hold great promise, but require careful characterisation by a number of approaches. Phage–bacteria infection time courses, where populations of bacteriophage and bacteria are mixed and followed over many infection cycles, can be used to deduce properties of phage infection at the individual cell level. Here, we apply this approach to analysis of infection of Escherichia coli by the temperate bacteriophage 186 and explore which properties of the infection process can be reliably inferred. By applying established modelling methods to such data, we extract the frequency at which phage 186 chooses the lysogenic pathway after infection, and show that lysogenisation increases in a graded manner with increased expression of the lysogenic establishment factor CII. The data also suggest that, like phage λ, the rate of lysogeny of phage 186 increases with multiple infections.Nan Hao, Dylan Agnew, Sandeep Krishna, Ian B. Dodd and Keith E. Shearwi

Gene Regulation in the Pi Calculus: Simulating Cooperativity at the Lambda Switch

Part of the Lecture Notes in Computer Science book series (LNCS, volume 4230).Also part of the Lecture Notes in Bioinformatics book sub series (volume 4230).International audienceWe propose to model the dynamics of gene regulatory networks as concurrent processes in the stochastic pi calculus. As a first case study, we show how to express the control of transcription initiation at the lambda switch, a prototypical example where cooperative enhancement is crucial. This requires concurrent programming techniques that are new to systems biology, and necessitates stochastic parameters that we derive from the literature. We test all components of our model by exhaustive stochastic simulations. A comparison with previous results reported in the literature, experimental and simulation based, confirms the appropriateness of our modeling approach

Instability of CII is needed for efficient switching between lytic and lysogenic development in bacteriophage 186

The CII protein of temperate coliphage 186, like the unrelated CII protein of phage λ, is a transcriptional activator that primes expression of the CI immunity repressor and is critical for efficient establishment of lysogeny. 186-CII is also highly unstable, and we show that in vivo degradation is mediated by both FtsH and RseP. We investigated the role of CII instability by constructing a 186 phage encoding a protease resistant CII. The stabilised-CII phage was defective in the lysis-lysogeny decision: choosing lysogeny with close to 100% frequency after infection, and forming prophages that were defective in entering lytic development after UV treatment. While lysogenic CI concentration was unaffected by CII stabilisation, lysogenic transcription and CI expression was elevated after UV. A stochastic model of the 186 network after infection indicated that an unstable CII allowed a rapid increase in CI expression without a large overshoot of the lysogenic level, suggesting that instability enables a decisive commitment to lysogeny with a rapid attainment of sensitivity to prophage induction.Iain M Murchland, Alexandra Ahlgren-Berg, Julian M J Pietsch, Alejandra Isabel, Ian B Dodd, Keith E Shearwi

Search for leptophobic Z ' bosons decaying into four-lepton final states in proton-proton collisions at root s=8 TeV

Peer reviewe

Searches for invisible decays of the Higgs boson in pp collisions at root S=7, 8, and 13 TeV

Peer reviewe

Measurements of differential production cross sections for a Z boson in association with jets in pp collisions at root s=8 TeV

Peer reviewe

Search for high-mass diphoton resonances in proton-proton collisions at 13 TeV and combination with 8 TeV search

Peer reviewe