The role of transcription-coupled DNA supercoiling in the coordination of temporal geneexpression.

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Understanding the concepts of genetic regulation and genome architecture is central for the design of artificial chromosomes for industrial purposes.We are facing the advent of a new era of transition from basic research in genetics to engineering.The technologies for sequence assembly are available, however the interplay of genes that was evolutionary optimized is still far from understood.Recent studies have shown that the position of a gene insertion into a native chromosome has a major impact on expression of the inserted gene as well as on the expression of the surrounding genes.Hence, for a de novo design of a chromosome it is important to understand the sources of such neighborhood dependencies.In this proposal we present striking preliminary data that reveals transcription-coupled DNA supercoiling (TCDS), a torsional tension introduced by transcribing RNA-polymerase, as a major source of positional effects.For individual genes it has been shown that TCDS affects gene expression of neighboring genes.We show evidence that the arrangement of genes on the whole E.coli chromosome is dictated by TCDS and the sensitivity of genes to TCDS.The regulatory concept of TCDS opens a new perspective to understand genetic flexibility in bacterial and viral pathogenicity or spontaneous drug resistance.Furthermore, it will allow for the construction of artificial regulatory circuits using particular gene arrangements.We therefore propose a comprehensive study of TCDS mediated regulation in the model organism E.coli.We plan to address the problem at different levels of organization.At a global level we will investigate the interdependencies of transcriptional activity of neighboring genes using state-of-the-art time-resolved gene expression data.With this data we will construct a TCDS network of local gene expression interdependencies and analyse its structure.Furthermore, we plan to integrate the TCDS network and the classical regulatory network consisting of links between regulators and their targets. Our preliminary data suggests a connection of the networks via the sensitivity of regulators and their targets to alterations in DNA supercoiling.For a deeper understanding of the local interactions of individual genes, we will study the expression of gene arrangements taken from the E.coli chromosome, alter its gene arrangement and compare the effects with our models.With the local and global approach we are convinced to elucidate the complexity of TCDS. As TCDS is caused by fundamental processes found in any organism we are highly optimistic to reveal important genetic insights far beyond the scope of the E.coli model organism and bacteria.