In growing bacterial cells, the global reorganization of transcription is associated with alterations of RNA polymerase composition and the superhelical density of the DNA. However, the existence of any regulatory device coordinating these changes remains elusive. Here we show that in an exponentially growing Escherichia coli rpoZ mutant lacking the polymerase ω subunit, the impact of the Eσ(38) holoenzyme on transcription is enhanced in parallel with overall DNA relaxation. Conversely, overproduction of σ(70) in an rpoZ mutant increases both overall DNA supercoiling and the transcription of genes utilizing high negative superhelicity. We further show that transcription driven by the Eσ(38) and Eσ(70) holoenzymes from cognate promoters induces distinct superhelical densities of plasmid DNA in vivo. We thus demonstrate a tight coupling between polymerase holoenzyme composition and the supercoiling regimen of genomic transcription. Accordingly, we identify functional clusters of genes with distinct σ factor and supercoiling preferences arranging alternative transcription programs sustaining bacterial exponential growth. We propose that structural coupling between DNA topology and holoenzyme composition provides a basic regulatory device for coordinating genome-wide transcription during bacterial growth and adaptation. IMPORTANCE Understanding the mechanisms of coordinated gene expression is pivotal for developing knowledge-based approaches to manipulating bacterial physiology, which is a problem of central importance for applications of biotechnology and medicine. This study explores the relationships between variations in the composition of the transcription machinery and chromosomal DNA topology and suggests a tight interdependence of these two variables as the major coordinating principle of gene regulation. The proposed structural coupling between the transcription machinery and DNA topology has evolutionary implications and suggests a new methodology for studying concerted alterations of gene expression during normal and pathogenic growth both in bacteria and in higher organisms.