DNA topology of highly transcribed operons in Salmonella enterica serovar Typhimurium

Abstract

SummaryBacteria differ from eukaryotes by having the enzyme DNA gyrase, which catalyses the ATP-dependent negative supercoiling of DNA. Negative supercoils are essential for condensing chromosomes into an interwound (plectonemic) and branched structure known as the nucleoid. Topo-1 removes excess supercoiling in an ATP-independent reaction and works with gyrase to establish a topological equilibrium where supercoils move within 10 kb domains bounded by stochastic barriers along the sequence. However, transcription changes the stochastic pattern by generating supercoil diffusion barriers near the sites of gene expression. Using supercoildependent Tn3 and gd resolution assays, we studied DNA topology upstream, downstream and across highly transcribed operons. Whenever two Res sites flanked efficiently transcribed genes, resolution was inhibited and the loss in recombination efficiency was proportional to transcription level. Ribosomal RNA operons have the highest transcription rates, and resolution assays at the rrnG and rrnH operons showed inhibitory levels 40-100 times those measured in low-transcription zones. Yet, immediately upstream and downstream of RNA polymerase (RNAP) initiation and termination sites, supercoiling characteristics were similar to poorly transcribed zones. We present a model that explains why RNAP blocks plectonemic supercoil movement in the transcribed track and suggests how gyrase and TopA control upstream and downstream transcriptiondriven supercoiling.

Large-Scale Conformational Transitions in Supercoiled DNA Revealed by Coarse-Grained Simulation

“…Similarly, recent experimental work in Caulobacter reveals that highly transcribed genes appear to demarcate topological domains in the chromosome (7), and in vivo measurements of interloci distances in Caulobacter resemble those theoretically predicted for a branched supercoiled conformation with densely packed plectonemes (65). In all three of these organisms, topological domains are measured to possess average sizes of~10 kilobases (7,33) and the importance of transcription in regulating the size of these topological domains is suggested by the high conservation of genomic interspacing of highly expressed genes in the chromosomes of phylogenetically distant bacteria (33). Thus, the linking deficits and length scales simulated in this work are of direct relevance to the length scales and linking deficit regimes that are implicated in transcriptional regulation of chromosomal topology, and may have important implications for chromosomal organization and dynamics.…”

“…Consistent with this model, both in vitro and in vivo studies indicate that RNA polymerase is a powerful torque generator, and linking deficits below s ¼ À0.10 have been measured as a result of transcription at strong promoters both in the presence of functional Topo I (DNA relaxing enzyme) as well as in Topo I-deficient mutants (28)(29)(30). Studies in E. coli and Salmonella have shown that RNA polymerase is capable of actively creating and remodeling dynamic supercoiled topological domains (6,33). Similarly, recent experimental work in Caulobacter reveals that highly transcribed genes appear to demarcate topological domains in the chromosome (7), and in vivo measurements of interloci distances in Caulobacter resemble those theoretically predicted for a branched supercoiled conformation with densely packed plectonemes (65).…”

“…A growing body of work reveals that regulation of DNA supercoiling in bacterial chromosomes entails an interplay between topoisomerases and transcription by RNA polymerase (6,10,(31)(32)(33). According to the twin supercoiled domain model (64), actively transcribing RNA polymerase must locally unwind the DNA double helix to provide access to the antisense strand.…”

“…Moreover, multiple lines of evidence indicate that RNA polymerase plays an active role, along with topoisomerases, in defining and remodeling topological domains in bacterial chromosomes (6,(31)(32)(33). Supercoiling, in turn, regulates transcription, leading to a complex interplay between chromosome topology and gene expression that is not fully understood.…”

DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression

“…The writhing of the underwound duplex is described as negative supercoiling (Higgins and Vologodskii 2015). In a bacterium such as Escherichia coli, about half of the DNA supercoils are constrained by interaction with proteins (Bliska and Cozzarelli 1987;Pettijohn and Pfenninger 1980), but the energy in the unconstrained portion is available to drive DNA transactions (Booker et al 2010). The constrained and unconstrained regions of the chromosome are unlikely to be fixed, displaying a dynamism that reflects changes in growth rate, growth phase and changes in the nature and sizes of the populations of DNA binding proteins that decorate the DNA.…”