Biomedical subjects

J Barry Egan

Publications and source records attributed to J Barry Egan.

9 recordsLinked to original sources

PubMed2006

The structural basis of cooperative regulation at an alternate genetic switch.

Bacteriophage lambda is a paradigm for understanding the role of cooperativity in gene regulation. Comparison of the regulatory regions of lambda and the unrelated temperate bacteriophage 186 provides insight into alternate ways to assemble functional genetic switches. The structure of the C-terminal domain of the 186 repressor, determined at 2.7 A resolution, reveals an unusual heptamer of dimers, consistent with presented genetic studies. In addition, the structure of a cooperativity mutant of the full-length 186 repressor, identified by genetic screens, was solved to 1.95 A resolution. These structures provide a molecular basis for understanding lysogenic regulation in 186. Whereas the overall fold of the 186 and lambda repressor monomers is remarkably similar, the way the two repressors cooperatively assemble is quite different and explains in part the differences in their regulatory activity.

Amino Acid Substitution↗

PubMed2005

Functional alignment of regulatory networks: a study of temperate phages.

The relationship between the design and functionality of molecular networks is now a key issue in biology. Comparison of regulatory networks performing similar tasks can provide insights into how network architecture is constrained by the functions it directs. Here, we discuss methods of network comparison based on network architecture and signaling logic. Introducing local and global signaling scores for the difference between two networks, we quantify similarities between evolutionarily closely and distantly related bacteriophages. Despite the large evolutionary separation between phage lambda and 186, their networks are found to be similar when difference is measured in terms of global signaling. We finally discuss how network alignment can be used to pinpoint protein similarities viewed from the network perspective.

Bacteriophages↗

PubMed2005

Revisited gene regulation in bacteriophage lambda.

The contribution of bacteriophage lambda to gene control research is far from over. A revised model of the lambda genetic switch includes extra cooperativity through octamerization of the cI repressor protein, mediated by long-range DNA looping. Structural analysis reveals remarkably subtle transcriptional activation by cI. The action of cI, activation by cII, and aspects of antitermination by N and Q all confirm the utility and versatility of simple, weak adhesive interactions mediated by nucleic acid tethers. New genetic and quantitative analysis of the lambda gene network is challenging cherished ideas about how complex behaviours emerge from this regulatory system.

Bacteriophage lambda↗

PubMed2005

Transcriptional interference--a crash course.

The term "transcriptional interference" (TI) is widely used but poorly defined in the literature. There are a variety of methods by which one can interfere with the process or the product of transcription but the term TI usually refers to the direct negative impact of one transcriptional activity on a second transcriptional activity in cis. Two recent studies, one examining Saccharomyces cerevisiae and the other Escherichia coli, clearly show TI at one promoter caused by the arrival of a transcribing complex initiating at a distant promoter. TI is potentially widespread throughout biology; therefore, it is timely to assess exactly its nature, significance and operative mechanisms. In this article, we will address the following questions: what is TI, how important and widespread is it, how does it work and where should we focus our future research efforts?

Escherichia coli↗

PubMed2004

A mathematical model for transcriptional interference by RNA polymerase traffic in Escherichia coli.

Interactions between RNA polymerases (RNAP) resulting from tandem or convergent arrangements of promoters can cause transcriptional interference, often with important consequences for gene expression. However, it is not known what factors determine the magnitude of interference and which mechanisms are likely to predominate in any situation. We therefore developed a mathematical model incorporating three mechanisms of transcriptional interference in bacteria: occlusion (in which passing RNAPs block access to the promoter), collisions between elongating RNAPs, and "sitting duck" interference (in which RNAP complexes waiting to fire at the promoter are removed by passing RNAP). The predictions of the model are in good agreement with a recent quantitative in vivo study of convergent promoters in E.coli. Our analysis predicts that strong occlusion requires the interfering promoter to be very strong. Collisions can also produce strong interference but only if the interfering promoter is very strong or if the convergent promoters are far apart (>200 bp). For moderate strength interfering promoters and short inter-promoter distances, strong interference is dependent on the sitting duck mechanism. Sitting duck interference is dependent on the relative strengths of the two promoters. However, it is also dependent on the "aspect ratio" (the relative rates of RNAP binding and firing) of the sensitive promoter, allowing promoters of equal strength to have very different sensitivities to transcriptional interference. The model provides a framework for using transcriptional interference to investigate various dynamic processes on DNA in vivo.

DNA-Directed RNA Polymerases↗

PubMed2004

Transcriptional interference between convergent promoters caused by elongation over the promoter.

Transcriptional interference with convergent transcription from face-to-face promoters is a potentially important form of gene regulation in all organisms. Using LacZ reporter studies, the mechanism of interference was determined for a pair of face-to-face prokaryotic promoters in which a strong promoter interferes 5.6-fold with a weak promoter, 62 bp away. The promoters were variously rearranged to test different models of interference. Terminating transcription from the strong promoter before it reached the weak promoter dramatically reduced interference, indicating a requirement for the passage of the converging RNAP over the weak promoter. Based on in vitro experiments showing a slow rate of escape for open complexes at the weak promoter and their sensitivity to head-on collisions with elongating RNAP, a "sitting duck" model of interference is proposed and supported with in vivo permanganate footprinting. The model is further supported by the analysis of a second set of prokaryotic face-to-face promoters.

Gene Expression Regulation, Bacterial↗

PubMed2004

Cooperativity in long-range gene regulation by the lambda CI repressor.

Effective repression of cI transcription from PRM by the bacteriophage lambda CI repressor requires binding sites (OL) located 2.4 kb from the promoter. A CI tetramer bound to OL1.OL2 interacts with a tetramer bound near PRM (OR1.OR2), looping the intervening DNA. We previously proposed that in this CI octamer:DNA complex, the distant OL3 operator and the weak OR3 operator overlapping PRM are juxtaposed so that a CI dimer at OL3 can cooperate with a CI dimer binding to OR3. Here we show that OL3 is necessary for effective repression of PRM and that the repressor at OL3 appears to interact specifically with the repressor at OR3. The OL3-CI-OR3 interaction involves the same CI interface used for short-range dimer-dimer interactions and does not occur without the other four operators. The long-range interactions were incorporated into a physicochemical model, allowing estimation of the long-range interaction energies and showing the lysogenic state to be ideally poised for CI negative autoregulation. The results establish the lambda system as a powerful tool for examining long-range gene regulatory interactions in vivo.

Binding Sites↗

PubMed2002

Action at a distance in CI repressor regulation of the bacteriophage 186 genetic switch.

The non-lambdoid coliphage 186 provides an alternative model to the lytic-lysogenic switch of phage lambda. Like lambda, the key switch regulator, the CI repressor, associates to octamers. Unlike lambda, the lytic promoter (pR) and the lysogenic promoter (pL) are face-to-face, 62 bp apart and are flanked by distal CI binding sites (FL and FR) located approximately 300 bp away. Using reporter and footprinting studies, we show that the outcome, but not the mechanism, of regulation by 186 CI is very similar to lambda. 186 CI stimulates pL transcription indirectly by repressing convergent interfering transcription from pR. However, in the absence of the flanking FL and FR sites, CI bound at pR interacts co-operatively with a weak CI binding site at pL and represses both promoters. FL and FR play a critical role; they assist repression of pR and simultaneously alleviate repression of pL, thus allowing high pL activity. We propose that the 186 switch is regulated by a novel mechanism in which a CI octamer bound at pR forms alternative DNA loops to pL or to a flanking site, depending on CI concentration.

Bacteriophage lambda↗

PubMed2001

The helix-turn-helix motif of the coliphage 186 immunity repressor binds to two distinct recognition sequences.

The CI protein of coliphage 186 is responsible for maintaining the stable lysogenic state. To do this CI must recognize two distinct DNA sequences, termed A type sites and B type sites. Here we investigate whether CI contains two separate DNA binding motifs or whether CI has one motif that recognizes two different operator sequences. Sequence alignment with 186-like repressors predicts an N-terminal helix-turn-helix (HTH) motif, albeit with poor homology to a large master set of such motifs. The domain structure of CI was investigated by linker insertion mutagenesis and limited proteolysis. CI consists of an N-terminal domain, which weakly dimerizes and binds both A and B type sequences, and a C-terminal domain, which associates to octamers but is unable to bind DNA. A fusion protein consisting of the 186 N-terminal domain and the phage lambda oligomerization domain binds A and B type sequences more efficiently than the isolated 186 CI N-terminal domain, hence the 186 C-terminal domain likely mediates oligomerization and cooperativity. Site-directed mutation of the putative 186 HTH motif eliminates binding to both A and B type sites, supporting the idea that binding to the two distinct DNA sequences is mediated by a variant HTH motif.

Amino Acid Sequence↗