Biochemical and genetic probing of transcriptional activity of the Escherichia coli chromosome.

摘要

DNA topoisomerases are ubiquitous enzymes playing an essential role in chromosome maintenance in all organisms. Enzymatic actions involved in transient DNA breaks affect nearly the biological processes using DNA as a template. These enzymes sustain a local change in DNA topology which has various biological effects including transcription. In Chapter 2 I assessed whole-genome mRNA abundances as a function of gene locations on the chromosome of Escherichia coli. Genomic and signal processing techniques allowed me to determine statistically significant patterns in the spatial series of transcriptional activities. Short- and long-range patterns of transcriptional activities correlate with the activities of a key DNA topoisomerase, DNA gyrase, and coincide with its distribution on the chromosome. The findings that spatial patterns of transcription can be modulated by changes in DNA supercoiling provide strong evidence of physiologically relevant higher-order organization of transcription in a bacterial chromosome. Perturbations of DNA gyrase can result in changes of DNA relaxation, DNA repair, and DNA replication. Each process affects complex transcriptional responses involving multiple genes and regulons. In Chapter 3, I carried out an analysis in which the individual effects on gene expression are independently collected and then combined within a linear model in order to understand the nature of a response. By representing the gyrase inhibition as a true pleiotropic phenomenon, I was able to demonstrate that: (i) DNA replication is required for the formation of spatial transcriptional domains; (ii) the transcriptional response to the gyrase inhibition is coordinated between at least two modules involved in DNA maintenance, relaxation and damage response; (iii) genes whose transcriptional response to gyrase inhibition does not depend on the main relaxation activity of the cell can be classified on the basis of a GC excess in their upstream and coding sequences; and (iv) relaxation by topoisomerase I dominates the transcriptional response followed by the effects of replication and RecA. Lastly, I investigated the distributions of DNA topoisomerase I, II, and IV on the E. coli chromosome using immunoprecipitation with antibodies specific against each topoisomerase and DNA microarray technique. Novel DNA-binding properties of DNA topoisomerases are discussed in Chapter 4.