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.Nan Hao, Kim Sneppen, Keith E. Shearwin, Ian B. Dod

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.Nan Hao, Keith E. Shearwin, Ian B. Dod

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.Nan Hao and Keith E. Shearwi

Single molecule analysis of DNA wrapping and looping by a circular 14mer wheel of the bacteriophage 186 CI repressor

The lytic–lysogenic decision in bacteriophage 186 is governed by the 186 CI repressor protein in a unique way. The 186 CI is proposed to form a wheel-like oligomer that can mediate either wrapped or looped nucleoprotein complexes to provide the cooperative and competitive interactions needed for regulation. Although consistent with structural, biochemical and gene expression data, many aspects of this model are based on inference. Here, we use atomic force microscopy (AFM) to reveal the various predicted wrapped and looped species, and new ones, for CI regulation of lytic and lysogenic transcription. Automated AFM analysis showed CI particles of the predicted dimensions on the DNA, with CI multimerization favoured by DNA binding. Measurement of the length of the wrapped DNA segments indicated that CI may move on the DNA, wrapping or releasing DNA on either side of the wheel. Tethered particle motion experiments were consistent with wrapping and looping of DNA by CI in solution, where in contrast to λ repressor, the looped species were exceptionally stable. The CI regulatory system provides an intriguing comparison with that of nucleosomes, which share the ability to wrap and release similar sized segments of DNA.Haowei Wang, Ian B. Dodd, David D. Dunlap, Keith E. Shearwin, and Laura Finz

The role of repressor kinetics in relief of transcriptional interference between convergent promoters

Transcriptional interference (TI), where transcription from a promoter is inhibited by the activity of other promoters in its vicinity on the same DNA, enables transcription factors to regulate a target promoter indirectly, inducing or relieving TI by controlling the interfering promoter. For convergent promoters, stochastic simulations indicate that relief of TI can be inhibited if the repressor at the interfering promoter has slow binding kinetics, making it either sensitive to frequent dislodgement by elongating RNA polymerases (RNAPs) from the target promoter, or able to be a strong roadblock to these RNAPs. In vivo measurements of relief of TI by CI or Cro repressors in the bacteriophage λ PR-PRE system show strong relief of TI and a lack of dislodgement and roadblocking effects, indicative of rapid CI and Cro binding kinetics. However, repression of the same λ promoter by a catalytically dead CRISPR Cas9 protein gave either compromised or no relief of TI depending on the orientation at which it binds DNA, consistent with dCas9 being a slow kinetics repressor. This analysis shows how the intrinsic properties of a repressor can be evolutionarily tuned to set the magnitude of relief of TI.Nan Hao, Adam C. Palmer, Alexandra Ahlgren-Berg, Keith E. Shearwin, and Ian B. Dod

Road rules for traffic on DNA - systematic analysis of transcriptional roadblocking in vivo

Genomic DNA is bound by many proteins that could potentially impede elongation of RNA polymerase (RNAP), but the factors determining the magnitude of transcriptional roadblocking in vivo are poorly understood. Through systematic experiments and modeling, we analyse how roadblocking by the lac repressor (LacI) in Escherichia coli cells is controlled by promoter firing rate, the concentration and affinity of the roadblocker protein, the transcription-coupled repair protein Mfd, and promoter-roadblock spacing. Increased readthrough of the roadblock at higher RNAP fluxes requires active dislodgement of LacI by multiple RNAPs. However, this RNAP cooperation effect occurs only for strong promoters because roadblock-paused RNAP is quickly terminated by Mfd. The results are most consistent with a single RNAP also sometimes dislodging LacI, though we cannot exclude the possibility that a single RNAP reads through by waiting for spontaneous LacI dissociation. Reducing the occupancy of the roadblock site by increasing the LacI off-rate (weakening the operator) increased dislodgement strongly, giving a stronger effect on readthrough than decreasing the LacI on-rate (decreasing LacI concentration). Thus, protein binding kinetics can be tuned to maintain site occupation while reducing detrimental roadblocking.Nan Hao, Sandeep Krishna, Alexandra Ahlgren-Berg, Erin E. Cutts, Keith E. Shearwin, and Ian B. Dod

“Genes”

In order to describe a cell at molecular level, a notion of a “gene” is neither necessary nor helpful. It is sufficient to consider the molecules (i.e., chromosomes, transcripts, proteins) and their interactions to describe cellular processes. The downside of the resulting high resolution is that it becomes very tedious to address features on the organismal and phenotypic levels with a language based on molecular terms. Looking for the missing link between biological disciplines dealing with different levels of biological organization, we suggest to return to the original intent behind the term “gene”. To this end, we propose to investigate whether a useful notion of “gene” can be constructed based on an underlying notion of function, and whether this can serve as the necessary link and embed the various distinct gene concepts of biological (sub)disciplines in a coherent theoretical framework. In reply to the Genon Theory recently put forward by Klaus Scherrer and Jürgen Jost in this journal, we shall discuss a general approach to assess a gene definition that should then be tested for its expressiveness and potential cross-disciplinary relevance

Cross-Mapping Events in miRNAs Reveal Potential miRNA-Mimics and Evolutionary Implications

MicroRNAs (miRNAs) have important roles in various biological processes. miRNA cross-mapping is a prevalent phenomenon where miRNA sequence originating from one genomic region is mapped to another location. To have a better understanding of this phenomenon in the human genome, we performed a detailed analysis in this paper using public miRNA high-throughput sequencing data and all known human miRNAs. We observed widespread cross-mapping events between miRNA precursors (pre-miRNAs), other non-coding RNAs (ncRNAs) and the opposite strands of pre-miRNAs by analyzing the high-throughput sequencing data. Computational analysis on all known human miRNAs also confirmed that many of them could be involved in cross-mapping events. The processing or decay of both ncRNAs and pre-miRNA opposite strand transcripts may contribute to miRNA enrichment, although some might be miRNA-mimics due to miRNA mis-annotation. Comparing to canonical miRNAs, miRNAs involved in cross-mapping events between pre-miRNAs and other ncRNAs normally had shorter lengths (17–19 nt), lower prediction scores and were classified as pseudo miRNA precursors. Notably, 4.9% of all human miRNAs could be accurately mapped to the opposite strands of pre-miRNAs, which showed that both strands of the same genomic region had the potential to produce mature miRNAs and simultaneously implied some potential miRNA precursors. We proposed that the cross-mapping events are more complex than we previously thought. Sequence similarity between other ncRNAs and pre-miRNAs and the specific stem-loop structures of pre-miRNAs may provide evolutionary implications

abd-A Regulation by the iab-8 Noncoding RNA

The homeotic genes in Drosophila melanogaster are aligned on the chromosome in the order of the body segments that they affect. The genes affecting the more posterior segments repress the more anterior genes. This posterior dominance rule must be qualified in the case of abdominal-A (abd-A) repression by Abdominal-B (Abd-B). Animals lacking Abd-B show ectopic expression of abd-A in the epidermis of the eighth abdominal segment, but not in the central nervous system. Repression in these neuronal cells is accomplished by a 92 kb noncoding RNA. This “iab-8 RNA” produces a micro RNA to repress abd-A, but also has a second, redundant repression mechanism that acts only “in cis.” Transcriptional interference with the abd-A promoter is the most likely mechanism

Bacteriophage Crosstalk: Coordination of Prophage Induction by Trans-Acting Antirepressors

Many species of bacteria harbor multiple prophages in their genomes. Prophages often carry genes that confer a selective advantage to the bacterium, typically during host colonization. Prophages can convert to infectious viruses through a process known as induction, which is relevant to the spread of bacterial virulence genes. The paradigm of prophage induction, as set by the phage Lambda model, sees the process initiated by the RecA-stimulated self-proteolysis of the phage repressor. Here we show that a large family of lambdoid prophages found in Salmonella genomes employs an alternative induction strategy. The repressors of these phages are not cleaved upon induction; rather, they are inactivated by the binding of small antirepressor proteins. Formation of the complex causes the repressor to dissociate from DNA. The antirepressor genes lie outside the immunity region and are under direct control of the LexA repressor, thus plugging prophage induction directly into the SOS response. GfoA and GfhA, the antirepressors of Salmonella prophages Gifsy-1 and Gifsy-3, each target both of these phages' repressors, GfoR and GfhR, even though the latter proteins recognize different operator sites and the two phages are heteroimmune. In contrast, the Gifsy-2 phage repressor, GtgR, is insensitive to GfoA and GfhA, but is inactivated by an antirepressor from the unrelated Fels-1 prophage (FsoA). This response is all the more surprising as FsoA is under the control of the Fels-1 repressor, not LexA, and plays no apparent role in Fels-1 induction, which occurs via a Lambda CI-like repressor cleavage mechanism. The ability of antirepressors to recognize non-cognate repressors allows coordination of induction of multiple prophages in polylysogenic strains. Identification of non-cleavable gfoR/gtgR homologues in a large variety of bacterial genomes (including most Escherichia coli genomes in the DNA database) suggests that antirepression-mediated induction is far more common than previously recognized