Coordination of genomic structure and transcription by the main bacterial nucleoid-associated protein HU

Abstract

The histone-like protein HU is a highly abundant DNA architectural protein that is involved in compacting the DNA of the bacterial nucleoid and in regulating the main DNA transactions, including gene transcription. However, the coordination of the genomic structure and function by HU is poorly understood. Here, we address this question by comparing transcript patterns and spatial distributions of RNA polymerase in Escherichia coli wild-type and hupA/B mutant cells. We demonstrate that, in mutant cells, upregulated genes are preferentially clustered in a large chromosomal domain comprising the ribosomal RNA operons organized on both sides of OriC. Furthermore, we show that, in parallel to this transcription asymmetry, mutant cells are also impaired in forming the transcription foci-spatially confined aggregations of RNA polymerase molecules transcribing strong ribosomal RNA operons. Our data thus implicate HU in coordinating the global genomic structure and function by regulating the spatial distribution of RNA polymerase in the nucleoid.

Figure 1

Spatial reorganization of transcription in cells lacking the histone-like protein HU. (A) Frequency distribution of gene transcripts upregulated in wild-type (grey) and hupA/B (black) backgrounds. (B) Frequency distributions of upregulated transcripts associated with DNA relaxation (grey) and high negative supercoiling (blue) in wild-type and hupA/B mutant cells (black and red, respectively). (C) Genomic distribution of gyrase-binding sites (Jeong et al, 2004). All frequency distributions were obtained by averaging, using a sliding window of 200 ORFs. Chromosomal positions of ribosomal RNA operons, OriC and Ter are indicated. (D) Dependence of cruciform formation (arrows) in (AT)₃₄ reporter plasmids on the superhelical density (σ) in vitro (left panel, lanes 1–8). The indicated σ values are within 15% of precision. Detection of cruciforms in exponentially growing wild-type and hupA/B mutant cells (right panel). 0.0, fully relaxed DNA template; oc, open circular form; ORFs, open reading frames; OriC, replication origin; rrn, ribosomal RNA operons; Ter, replication terminus; wt, wild type.

Figure 2

Exponentially growing hupA/B mutant cells are impaired in foci formation. (A) Representative fluorescence microscopy images of wild-type cells showing RNAP-dense areas (foci) at the poles of the nucleoid (white arrowheads; only two positions are indicated). (B) Representative image of a hupA/B mutant cell. It should be noted that RNAP is more evenly distributed in the nucleoid. Fluorescence images of RpoC-YFP (RNAP), DRAQ5 (nucleoid) and phase-contrast images are shown. In the merged images, green is the RpoC-YFP fluorescence signal, and red is the DRAQ5 fluorescence signal. Scale bars, 2 μm. RNAP, RNA polymerase; YFP, yellow fluorescent protein.

Figure 3

Transcription foci localize near OriC at opposite poles of the nucleoid. (A) Fluorescence microscopy image of wild-type cells carrying the rpoC-yfp fusion allele and 240 tandem lac operator insertions at position 3,908 kb close to OriC (Lau et al, 2003). Fluorescence images of RpoC-YFP (RNAP), LacI-CFP (OriC), DRAQ5 (nucleoid) and phase-contrast images are shown in greyscale on the left. Merge images are shown on the right: green is the RpoC-YFP fluorescence signal; red is LacI-CFP; and blue is DRAQ5 fluorescence signal. Scale bar, 2 μm. (B) Images of wild-type cells carrying the rpoC-yfp fusion allele and 240 tandem lac operator insertions at position 1,801 kb close to Ter. CFP, cyan fluorescent protein; RNAP, RNA polymerase; Ter, replication terminus; YFP, yellow fluorescent protein.

Figure 4

Domain organization of the genome. (A) Approximate spatial arrangement of the rrn macrodomain with respect to the chromosomal macrodomains determined by Valens et al (2004). (B) A model of nucleoid structure organization by HU. At stable RNA operons, the negative twist introduced into the DNA by more than 50 transcribing polymerases (French & Miller, 1989) constrains, in a closed topological domain of about 5 kb in size, a high negative superhelical density (at −σ=∼0.1) with compensatory increase in positive superhelicity. Constraint of these positive supercoils (+) by HU acts as a ‘topological sink' buffering the diffusion of positive superhelicity and stabilizing transcription (left panel). In the absence of HU, the increased accessibility of gyrase-binding sites in the rrn macrodomain imposes an imbalance on supercoil distribution and asymmetry on genomic transcription (right side). NS, non-structured macrodomains; rrn, ribosomal RNA; Ter, replication terminus; wt, wild type.