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.

Efficient O-demethylation of lignin monoaromatics using the peroxygenase activity of cytochrome P450 enzymes.

A crucial reaction in harnessing renewable carbon from lignin is O-demethylation. We demonstrate the selective O-demethylation of syringol and guaiacol using different cytochrome P450 enzymes. These can efficiently use hydrogen peroxide which, when compared to nicotinamide cofactor-dependent monooxygenases and synthetic methods, allows for cheap and clean O-demethylation of lignin-derived aromatics.

Analysis of Infection Time Courses Shows CII Levels Determine the Frequency of Lysogeny in Phage 186.

Engineered 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.

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

We 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.

Simplified heavy-atom derivatization of protein structures via co-crystallization with the MAD tetragon tetrabromoterephthalic acid.

The phase problem is a persistent bottleneck that impedes the structure-determination pipeline and must be solved to obtain atomic resolution crystal structures of macromolecules. Although molecular replacement has become the predominant method of solving the phase problem, many scenarios still exist in which experimental phasing is needed. Here, a proof-of-concept study is presented that shows the efficacy of using tetrabromoterephthalic acid (B4C) as an experimental phasing compound. Incorporating B4C into the crystal lattice using co-crystallization, the crystal structure of hen egg-white lysozyme was solved using MAD phasing. The strong anomalous signal generated by its four Br atoms coupled with its compatibility with commonly used crystallization reagents render B4C an effective experimental phasing compound that can be used to overcome the phase problem.

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

The loopometer: a quantitative in vivo assay for DNA-looping proteins.

Proteins that can bring together separate DNA sites, either on the same or on different DNA molecules, are critical for a variety of DNA-based processes. However, there are no general and technically simple assays to detect proteins capable of DNA looping in vivo nor to quantitate their in vivo looping efficiency. Here, we develop a quantitative in vivo assay for DNA-looping proteins in Escherichia coli that requires only basic DNA cloning techniques and a LacZ assay. The assay is based on loop assistance, where two binding sites for the candidate looping protein are inserted internally to a pair of operators for the E. coli LacI repressor. DNA looping between the sites shortens the effective distance between the lac operators, increasing LacI looping and strengthening its repression of a lacZ reporter gene. Analysis based on a general model for loop assistance enables quantitation of the strength of looping conferred by the protein and its binding sites. We use this 'loopometer' assay to measure DNA looping for a variety of bacterial and phage proteins.

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.

A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186.

The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

Advanced Resistance Studies Identify Two Discrete Mechanisms in Staphylococcus aureus to Overcome Antibacterial Compounds that Target Biotin Protein Ligase.

Biotin protein ligase (BPL) inhibitors are a novel class of antibacterial that target clinically important methicillin-resistant Staphylococcus aureus (S. aureus). In S. aureus, BPL is a bifunctional protein responsible for enzymatic biotinylation of two biotin-dependent enzymes, as well as serving as a transcriptional repressor that controls biotin synthesis and import. In this report, we investigate the mechanisms of action and resistance for a potent anti-BPL, an antibacterial compound, biotinyl-acylsulfamide adenosine (BASA). We show that BASA acts by both inhibiting the enzymatic activity of BPL in vitro, as well as functioning as a transcription co-repressor. A low spontaneous resistance rate was measured for the compound (<10-9) and whole-genome sequencing of strains evolved during serial passaging in the presence of BASA identified two discrete resistance mechanisms. In the first, deletion of the biotin-dependent enzyme pyruvate carboxylase is proposed to prioritize the utilization of bioavailable biotin for the essential enzyme acetyl-CoA carboxylase. In the second, a D200E missense mutation in BPL reduced DNA binding in vitro and transcriptional repression in vivo. We propose that this second resistance mechanism promotes bioavailability of biotin by derepressing its synthesis and import, such that free biotin may outcompete the inhibitor for binding BPL. This study provides new insights into the molecular mechanisms governing antibacterial activity and resistance of BPL inhibitors in S. aureus.

Combining random microseed matrix screening and the magic triangle for the efficient structure solution of a potential lysin from bacteriophage P68.

Two commonly encountered bottlenecks in the structure determination of a protein by X-ray crystallography are screening for conditions that give high-quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5-amino-2,4,6-triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high-quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg-white lysozyme and the N-terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion-associated lysin or endolysin.

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.

Characterizing Transcriptional Interference between Converging Genes in Bacteria.

Antisense transcription is common in naturally occurring genomes and is increasingly being used in synthetic genetic circuitry as a tool for gene expression control. Mutual influence on the expression of convergent genes can be mediated by antisense RNA effects and by transcriptional interference (TI). We aimed to quantitatively characterize long-range TI between convergent genes with untranslated intergenic spacers of increasing length. After controlling for antisense RNA-mediated effects, which contributed about half of the observed total expression inhibition, the TI effect was modeled. To achieve model convergence, RNA polymerase processivity and collision resistance were assumed to be modulated by ribosome trailing. The spontaneous transcription termination rate in regions of untranslated DNA was experimentally determined. Our modeling suggests that an elongating RNA polymerase with a trailing ribosome is about 13 times more likely to resume transcription than an opposing RNA polymerase without a trailing ribosome, upon head-on collision of the two.

Native mass spectrometry identifies an alternative DNA-binding pathway for BirA from Staphylococcus aureus.

An adequate supply of biotin is vital for the survival and pathogenesis of Staphylococcus aureus. The key protein responsible for maintaining biotin homeostasis in bacteria is the biotin retention protein A (BirA, also known as biotin protein ligase). BirA is a bi-functional protein that serves both as a ligase to catalyse the biotinylation of important metabolic enzymes, as well as a transcriptional repressor that regulates biotin biosynthesis, biotin transport and fatty acid elongation. The mechanism of BirA regulated transcription has been extensively characterized in Escherichia coli, but less so in other bacteria. Biotin-induced homodimerization of E. coli BirA (EcBirA) is a necessary prerequisite for stable DNA binding and transcriptional repression. Here, we employ a combination of native mass spectrometry, in vivo gene expression assays, site-directed mutagenesis and electrophoretic mobility shift assays to elucidate the DNA binding pathway for S. aureus BirA (SaBirA). We identify a mechanism that differs from that of EcBirA, wherein SaBirA is competent to bind DNA as a monomer both in the presence and absence of biotin and/or MgATP, allowing homodimerization on the DNA. Bioinformatic analysis demonstrated the SaBirA sequence used here is highly conserved amongst other S. aureus strains, implying this DNA-binding mechanism is widely employed.

Positive and Negative Control of Enhancer-Promoter Interactions by Other DNA Loops Generates Specificity and Tunability.

Enhancers are ubiquitous and critical gene-regulatory elements. However, quantitative understanding of the role of DNA looping in the regulation of enhancer action and specificity is limited. We used the Escherichia coli NtrC enhancer-σ54 promoter system as an in vivo model, finding that NtrC activation is highly sensitive to the enhancer-promoter (E-P) distance in the 300-6,000 bp range. DNA loops formed by Lac repressor were able to strongly regulate enhancer action either positively or negatively, recapitulating promoter targeting and insulation. A single LacI loop combining targeting and insulation produced a strong shift in specificity for enhancer choice between two σ54 promoters. A combined kinetic-thermodynamic model was used to quantify the effect of DNA-looping interactions on promoter activity and revealed that sensitivity to E-P distance and to control by other loops is itself dependent on enhancer and promoter parameters that may be subject to regulation.

Biotin-mediated growth and gene expression in Staphylococcus aureus is highly responsive to environmental biotin.

Biotin (Vitamin B7) is a critical enzyme co-factor in metabolic pathways important for bacterial survival. Biotin is obtained either from the environment or by de novo synthesis, with some bacteria capable of both. In certain species, the bifunctional protein BirA plays a key role in biotin homeostasis as it regulates expression of biotin biosynthetic enzymes in response to biotin demand and supply. Here, we compare the effect of biotin on the growth of two bacteria that possess a bifunctional BirA, namely Escherichia coli and Staphylococcus aureus. Unlike E. coli that could fulfill its biotin requirements through de novo synthesis, S. aureus showed improved growth rates in media supplemented with 10 nM biotin. S. aureus also accumulated more radiolabeled biotin from the media highlighting its ability to efficiently scavenge exogenous material. These data are consistent with S. aureus colonizing low biotin microhabitats. We also demonstrate that the S. aureus BirA protein is a transcriptional repressor of BioY, a subunit of the biotin transporter, and an operon containing yhfT and yhfS, the products of which have a putative role in fatty acid homeostasis. Increased expression of bioY is proposed to help cue S. aureus for efficient scavenging in low biotin environments.

Programmable DNA looping using engineered bivalent dCas9 complexes.

DNA looping is a ubiquitous and critical feature of gene regulation. Although DNA looping can be efficiently detected, tools to readily manipulate DNA looping are limited. Here we develop CRISPR-based DNA looping reagents for creation of programmable DNA loops. Cleavage-defective Cas9 proteins of different specificity are linked by heterodimerization or translational fusion to create bivalent complexes able to link two separate DNA regions. After model-directed optimization, the reagents are validated using a quantitative DNA looping assay in E. coli. Looping efficiency is ~15% for a 4.7 kb loop, but is significantly improved by loop multiplexing with additional guides. Bivalent dCas9 complexes are also used to activate endogenous norVW genes by rewiring chromosomal DNA to bring distal enhancer elements to the gene promoters. Such reagents should allow manipulation of DNA looping in a variety of cell types, aiding understanding of endogenous loops and enabling creation of new regulatory connections.

Hidden Secrets of Sigma54 Promoters Revealed.

Bacterial sigma54 (σ54) promoters are the DNA-binding motif for σ54-containing RNA polymerase (RNAP) holoenzymes. A recent study using a combination of synthetic oligonucleotide library screening, biochemical characterization, and bioinformatics has uncovered a new and unexpected role for σ54 promoters, encoding a form of bacterial 'insulator sequence' to dampen unwanted translation.

Efficient chromosomal-scale DNA looping in Escherichia coli using multiple DNA-looping elements.

Genes are frequently regulated by interactions between proteins that bind to the DNA near the gene and proteins that bind to DNA sites located far away, with the intervening DNA looped out. But it is not understood how efficient looping can occur when the sites are very far apart. We develop a simple theoretical framework that relates looping efficiency to the energetic cost and benefit of looping, allowing prediction of the efficiency of single or multiple nested loops at different distances. Measurements of absolute loop efficiencies for Lac repressor and λ CI using gene expression reporters in Escherichia coli cells show that, as predicted by the model, long-range DNA looping between a pair of sites can be strongly enhanced by the use of nested DNA loops or by the use of additional protein-binding sequences. A combination of these approaches was able to generate efficient DNA looping at a 200 kb distance.

Directing traffic on DNA-How transcription factors relieve or induce transcriptional interference.

Transcriptional interference (TI) is increasingly recognized as a widespread mechanism of gene control, particularly given the pervasive nature of transcription, both sense and antisense, across all kingdoms of life. Here, we discuss how transcription factor binding kinetics strongly influence the ability of a transcription factor to relieve or induce TI.